Cerus Corporation INTERCEPT Blood System for plasma

Transcription

STRAMER ET AL.
TABLE 3. Log10 reduction of bacteria and spirochetes in platelets
INTERCEPT71
Gram Negative
Escherichia coli
Serratia marcescens
Klebsiella pneumoniae
Pseudomonas aeruginosa
Salmonella choleraesuis
Yersinia enterocolitica
Enterobacter cloacae
Gram Positive
Staphylococcus epidermidis
S. aureus
S. aureus MRSA Strain
Streptococcus pyogenes
Listeria monocytogenes
Corynebacterium minutissimum
Bacillus cereus (includes spores)
B. cereus (vegetative)
B. cereus (isolated from donated blood)
Bifidobacterium adolescentis
Propionibacterium acnes
Lactobacillus species
Clostridium perfringens (vegetative form)
Spirochetes
Treponema pallidum
Borrelia burgdorferi
>6.4
>6.7
>5.6
4.5
>6.2
>5.9
5.9
Mirasol66
>4.4
4.0
>4.5, >4.7*
>6.6
6.6
4.2
3.6
4.8
>6.8
>6.3
>6.3
3.6
>5.5
1.9†
2.7
>6.0
>6.2
>6.4
>6.5
4.8
ⱖ4.8
4.3
4.5
ⱖ4.7
ⱖ6.8 to ⱕ7.0
>6.8
Cerus Corporation INTERCEPT
Blood System for plasma
The INTERCEPT system for plasma is
similar to their platelet system. After
addition of amotosalen to the plasma
and illumination, the plasma flows
through a Compound Adsorption Device (CAD) to remove
unreacted amotosalen and photoproducts. The process is
shown in Fig. 5.
TABLE 4. Log10 reduction of protozoa/rickettsia
in platelets
INTERCEPT
ⱖ6.072
>5.373,74
>5.076
>4.376
>5.372
ⱖ4.0
ⱖ5.0
4.8
ⱖ4.9
ⱖ4.3
* ATCC 43088 and ATCC 27853, respectively.
† ATCC 7064.
Plasmodium falciparum
Trypanosoma cruzi
Leishmania mexicana
Leishmania major, strain Jish
Babesia microti
Orientia tsutsugamushi
Theraflex
UV65
their respective platelet PR system
outlined above with some minor modifications. MacoPharma (Theraflex©
MB-plasma) has developed a photochemical process that incorporates
methylene blue (MB) and visible light.
None is approved for clinical use in the
US or Canada. These processes are
shown in Figs. 5 through 7, respectively.
Octapharma (Lachen, Switzerland;
http://www.octapharma.com) developed a PR process for pooled plasma
units intended for large-scale manufacturing of what is commonly known as
solvent/detergent (S/D) plasma (Octaplas).79,80 A slightly different S/D plasma
product manufactured by Vitex was
licensed in the US but is no longer
marketed.79,81,82
Mirasol
6.075
CaridianBCT Biotechnologies Mirasol plasma
system
>5.077
CaridianBCT Biotechnologies plasma system requires a
transfer of the plasma into a final freezing/storage container after illumination. This process is shown in Fig. 6.
>5.078
MacoPharma Theraflex MB-plasma system
MacoPharma’s Theraflex MB requires 1 mM MB and
180 J/cm2 illumination dose using low-pressure sodium
lights with a peak wavelength of 590 nm for inactivation.
Since MB cannot permeate leukocytes, the Theraflex MB
disposable is offered in two formats: if the plasma has
already been membrane filtered, the set does not include
a leukoreduction filter. Alternatively, if the plasma has
not been leukoreduced, the disposable incorporates a
leukoreduction filter. Both configurations include a filter
for removal of MB and photoproducts.84 This process is
shown in Fig. 7.
Octapharma octaplas® plasma system
S/D treatment of plasma is performed by adding 1% (w/w)
tri(n-butyl) phosphate (TNBP) and 1% (w/w) octoxynol-9
Fig. 5. Cerus Corporation INTERCEPT Blood System for
plasma.83
18S TRANSFUSION
Volume 49, August 2009 Supplement
297
EID AND TRANSFUSION SAFETY
resistant to the S/D inactivation process
or if neutralizing antibody is not present
at a sufficient titer to neutralize the challenging agent.
Pathogen reduction in plasma
Published results for pathogen reduction in plasma are provided in Tables 5
through 8. Data from abstracts or
Fig. 6. CaridianBCT Biotechnologies Mirasol plasma system.
unpublished data available from studies
conducted by the manufacturers are
included as personal communications
from the companies’ representatives.
Cerus (INTERCEPT) has demonstrated
inactivation of >5.5 logs of O. tsutsugamushi in an animal model. CaridianBCT
Biotechnologies (Mirasol) has evaluated
inactivation of a variety of transfusiontransmitted and model viruses (enveloped and nonenveloped) and parasites.
The inactivation spectrum in plasma is
the same as Mirasol platelets, since the
platelets are suspended in 100% plasma
and plasma actually constitutes the bulk
Fig. 7. MacoPharma Theraflex MB-plasma system.
of the media in which the inactivation
process is being performed (Raymond
TABLE 5. Log10 pathogen reduction for known or potential
Goodrich, pers. comm., 2009). Octaptransfusion-transmitted viruses in plasma
harma has conducted additional recent
Virus
INTERCEPT83
Theraflex MB85,86
Octaplas81,82
viral inactivation studies that have
Enveloped
HIV-1
shown: HIV-1 (>6.3 logs); VSV (ⱖ7.5
• Cell-free
>6.8
ⱖ5.45
ⱖ7.2
logs); Sindbis (>5.4 logs); pseudorabies
• Cell-associated
>6.4
virus (>6.3 logs); and Herpes simplex
ⱖ6.0 CID50
HBV
>4.5 CID50*
HCV
>4.5 CID50
ⱖ5.0 CID50
virus-2 (>6.1 logs). Since the product is
HTLV-I, cell-associated
ⱖ4.5
cell free, risk from cell-associated
HTLV-II, cell-associated
>5.7
viruses is also reduced. Further stuCMV
ⱖ4.08
WNV
ⱖ6.8
ⱖ5.78
dies have demonstrated a substantial
CHIKV
ⱖ7.6
immune neutralization capacity in
Nonenveloped
plasma pools for the following viruses:
B19V
1.8
HAV
0†
B19V (10.8 logs); HAV (ⱖ10.0 logs); HEV
* Chimp Infectious Dose where 50% of the animals become infected.
(ⱖ9.4 logs); Coxsackievirus B6 (ⱖ8.6
† No reduction was observed.
logs); HSV-1 (>11.1 logs) and poliovirus
type 1 (ⱖ10.9 logs). (Tor-Einar Svae
and Marc Maltas, pers. comm., 2009);
Immune neutralization occurs when an antibody specific
(Triton X-100) to plasma and incubating for 4 hours at
for an infectious agent binds to that agent and renders it
30°C. The plasma is then subjected to oil extraction and
noninfectious, but such neutralization cannot always be
phase separation to remove the TNBP; solid phase extracrelied on because of the unknown prevalence of specific
tion to remove the Triton X-100; sterile filtration and
antibodies in the donor population.
aseptic filling into plastic containers; and fast freezing at
ⱕ-60°C, followed by storage at ⱕ-30°C.80 It should be
noted that S/D treatment of plasma is not intended as a PR
RED BLOOD CELL PATHOGEN REDUCTION
method for nonenveloped viruses. S/D plasma is manuSYSTEMS
factured by pooling approximately 600-1500 source or
recovered plasma donations, respectively; it is possible
CaridianBCT Biotechnologies is using a photochemical
that transmission of an agent may occur if that agent is
process for treating whole blood which incorporates
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STRAMER ET AL.
TABLE 6. Log10 reduction of model viruses in plasma
Virus
Enveloped
Vesicular stomatitis
Influenza A (H5N1)
Duck HBV
Bovine viral diarrhea
Sindbis
Pseudorabies virus
Infectious bronchitis virus
Herpes simplex virus-2
SARS
Nonenveloped
Bluetongue
Calicivirus
Human adenovirus 5
Model for
Enveloped viruses
Influenza A viruses
HBV
HCV
HCV
CMV, HSV
Coronaviruses
Enveloped viruses
>5.7
4.4-4.5
ⱖ6.0
>4.9
ⱖ4.4
ⱖ6
ⱖ5.44
Nonenveloped viruses
Gram Negative
Klebsiella pneumoniae
Yersinia enterocolitica
Anaplasma phagocytophilum (HGA) agent
Gram Positive
Staphylococcus epidermidis
Spirochetes
Treponema pallidum
Borrelia burgdorferi
ⱖ6.8
ⱖ7.3 ID50
ⱖ6.1
ⱖ6.9
ⱖ7
ⱖ3.9
ⱖ5.33
requiring platelet transfusion support. In the US, Cerus
also conducted radiolabel recovery and survival studies in
healthy volunteers, including a study in which treated
platelets were also gamma irradiated.88 Results from these
studies showed a 15-20% decrease in radiolabel recovery
and survival studies in INTERCEPT platelets compared to
control platelets. One of the patient trials was a bleeding
time study. The bleeding time correction and time to next
transfusion were not statistically different between the
groups, despite a lower corrected count increment (CCI)
in the INTERCEPT group.89
INTERCEPT platelets were evaluated in thrombocytopenic patients in a two-arm, double-blind clinical Phase
III-like trial in Europe using buffy coat platelets (euroSPRITE).90 One hundred three (103) subjects were enrolled
in this trial, 52 patients received 311 test transfusions, and
51 patients received 256 control transfusions. Patients
who received test platelets showed no statistically significant differences in their CCI compared to controls at 1
hour; however, the CCIs at 24 hours did become significantly different. There was an insignificant decrease in
time between transfusions (3.0 vs. 3.4 days, test vs. control,
respectively). Clinical hemostasis, hemorrhagic, and
aggregate adverse events were similar between groups.
Cerus also conducted a smaller pilot study in Europe that
showed that 7-day INTERCEPT platelets were well tolerated and prevented bleeding. Noninferiority in terms of
1-hour CCI could not be demonstrated with a prespecified
noninferiority margin of 2200.91
In the US, Cerus conducted a large (n = 645) Phase
III double-blind, two-arm trial (INTERCEPT vs. untreated
platelets) in thrombocytopenic patients using apheresis
platelets (SPRINT). Clinical efficacy as measured by incidence of WHO bleeding Grade 2, 3, and 4 was not
different between the two groups. However, 1-hour CCI,
days to the next platelet transfusion, and number of
platelet transfusions were statistically different, all favoring the control group.92 This finding reflected a difference
in the mean platelet dose per transfusion in the two
ⱖ7.4
>7.3
>4.2
>7.3
>5.9
>10.6
Theraflex MB
>4.9 to >5.887
riboflavin and UV light that is similar to their Mirasol
process for platelets and plasma. This product is currently
being tested in clinical trials in the US under an FDAapproved investigational device exemption (IDE)
(Raymond Goodrich, pers. comm., 2009).
Cerus Corporation is designing a process for RBCs
that uses a chemical cross-linker, specific for nucleic acid.
Cerus’ product entered a Phase III trial several years ago in
the US, but issues with neoantigen formation brought that
trial to a halt. A redesign of the process has eliminated
immunogenicity in laboratory and animal studies. A
Phase I trial in healthy volunteers is underway (Lily Lin,
pers. comm., 2009).
CLINICAL TRIALS/CLINICAL EXPERIENCE
Platelet clinical trials
Cerus Corporation INTERCEPT platelets have undergone
examination in at least 4 clinical trials in patients
ⱖ8
5.1
TABLE 8. Log10 reduction of protozoa in plasma
INTERCEPT83
ⱖ6.9
>5.0
>5.3
Octaplas81,82
>6.1
ⱖ5.5
INTERCEPT83
20S TRANSFUSION
Theraflex MB85,86
ⱖ5.48
4.9
TABLE 7. Log10 reduction of bacteria and
spirochetes in plasma
Plasmodium falciparum
Trypanosoma cruzi
Babesia microti
INTERCEPT83
299
transfusion-associated graft-versus-host disease (TAGVHD) were reported. Furthermore, the use of 65% additive solution to replace plasma in this platelet PR system
may have contributed to a lower rate of acute transfusion
reactions.97,98 In a study completed in 2006 at the Blood
Transfusion Center, Mont Godinne, Belgium, blood component usage was evaluated in two 3-year blocks, one
before implementation of INTERCEPT and one after, in
688 and 795 patients, respectively. Primary diagnoses
were similar in the two study groups: hematology
(approximately 40%), oncology (approximately 6%), cardiovascular surgery (approximately 30%), and other
surgery and general medical services (approximately
20%). Red cell and platelet usage was statistically unchanged before and after implementation of
INTERCEPT.99
In a separate study, the routine use experience from
two periods in EFS-Alsace, France was compared, one
before implementation of platelet additive solution and
INTERCEPT and one after, in 2050 and 2069 patients,
respectively. In both periods, patients were transfused
according to conventional medical indications. The
results show that transfusion of INTERCEPT platelets to a
broad patient population for a spectrum of indications
was well tolerated. The incidence of adverse events was
less than untreated platelet components suspended in
plasma. No increase in the total platelet dose and RBCs
transfused to patients was observed.100
Two additional clinical studies were conducted per
country specific requirements: one in Luebeck, Germany101 and the other in Basel, Switzerland.102 Physicians
ordered INTERCEPT platelets according to standard clinical practice. However, INTERCEPT platelets were used in
place of gamma irradiation for prevention of TA-GVHD.
In the Luebeck study, 560 INTERCEPT transfusions were
administered to 52 patients with hematological malignancies. In the Basel study, 551 INTERCEPT platelet components were administered to 46 patients of whom 38
were hematology-oncology patients. The results of these
studies show low rates of overall acute platelet transfusion reactions. No bleeding complications were attributable to the INTERCEPT platelets. Results of an
investigator study involving 500 INTERCEPT platelet
transfusions in 83 pediatric hematology-oncology
patients in a routine clinical setting were reported.103 This
study showed that transfusion of pediatric patients with
INTERCEPT platelets was well tolerated and provided
therapeutic count increments.
As part of CaridianBCT Biotechnologies Mirasol
Evaluation Program, several hundred Mirasol-treated
platelet products have been transfused in routine use. No
Mirasol-related adverse reactions have been reported at
any of the participating blood centers or hospital sites.
This program has been expanded considerably in 2009
and is targeting several thousand transfusions which will
groups; the INTERCEPT group patients received a higher
proportion of doses <3.0 ¥ 1011. There was a statistically
significant reduction in transfusion reactions in those
patients receiving the INTERCEPT platelets compared to
the control (3 vs. 4.1%). However, there was an increase
in three specific pulmonary events in the INTERCEPT
group: acute respiratory distress syndrome (ARDS),
pneumonitis not otherwise specified, and pleuritic chest
pain. Reanalysis of this apparent increase of pulmonary
adverse reactions with INTERCEPT transfusions by both
the study investigators and independent experts blinded
to patient treatment did not confirm this difference. The
original observations were attributed to inconsistent
reporting of ARDS from SPRINT study sites and characteristics of the classification system used.93,94
CaridianBCT Biotechnologies Mirasol Platelets have
been tested in three clinical trials. The company studied
in vivo recovery and survival of platelets in normal volunteers and found an approximate 25% decrease in
recovery and survival in the Mirasol group.95 Results of a
Phase III-like clinical trial at six blood establishments
and six hospitals in France were reported at the 2008
AABB Annual Meeting.96 In this study, efficacy data were
analyzed on 80 subjects who received Mirasol-treated
(test) or control apheresis platelets. Patients who
received test platelets showed a statistically significant
(p < 0.002) lower 1-hour CCI compared to control platelets. However, the CCIs were not statistically different at
24 hours. There was also a statistically shorter time
between transfusions during the period of the first eight
transfusions in the test group compared to the control;
this difference was not observed beyond the eighth
transfusion. The number of platelet transfusions per
patient, total platelet dose, platelet transfusion per day of
support, percent refractory patients, the number of red
cells per patient, the number of WHO Grade 2, 3 and 4
bleeding events, and serious adverse events were not statistically different between the groups (p > 0.05). No
neoantigenicity was observed.
MacoPharma has completed preclinical studies of its
Theraflex UV process; it is currently under evaluation in
Phase I studies.
Platelet clinical experience
To date, over 200,000 Cerus INTERCEPT platelet doses
have been transfused in routine clinical use. An ongoing
postmarketing observational hemovigilance program has
been established to monitor the safety profile of INTERCEPT platelets. Two reports representing over 12,500
transfusions of INTERCEPT platelets to 2051 patients in
11 European centers in five countries from October 2003
to January 2007 demonstrated good clinical tolerance
and a safety profile similar to untreated platelets. No
episodes of transfusion-related acute lung injury or
300
TRANSFUSION 21S
STRAMER ET AL.
Octapharma octaplas has been evaluated in several
uncontrolled, observational trials in liver transplant,
cardiac surgery, and TTP patients, with no differences
noted between S/D plasma and FFP. There have been a
few randomized, controlled clinical trials in patients with
severe coagulopathy or undergoing cardiopulmonary
bypass surgery, with no clinical differences noted.
Whether these studies were sufficiently powered to see
any differences is debatable.
be monitored for adverse event reporting (Raymond Goodrich, pers. comm., 2009).
Plasma clinical trials
INTERCEPT plasma has been evaluated in clinical studies
in the US. In their first human clinical study, subjects
donated plasma with half of the plasma treated with the
INTERCEPT system and half prepared as standard fresh
frozen plasma (FFP). Subjects then received warfarin over
4 days to lower the Factor VII levels. On day 4, subjects
received either their own standard FFP or INTERCEPTFFP. After 2 weeks, subjects underwent an identical protocol and received the other type of FFP. Factor VII kinetics
were the same in subjects after either INTERCEPT FFP or
standard FFP.104 The efficacy and safety of INTERCEPT
plasma in patients with congenital coagulation factor
deficiencies was evaluated in a single-arm open-label
clinical trial. The results of this 34-patient trial with deficiencies of coagulation factors I (fibrinogen), II, V, VII, X,
XI, and XIII demonstrated that INTERCEPT plasma provided coagulation factor recovery and pharmacokinetics
comparable to conventional plasma, with prothrombin
time (PT) and activated partial thromboplastin time
(aPTT) responses sufficient for adequate hemostasis.105
Furthermore, in a randomized, double-blind clinical trial
in 121 patents with acquired coagulopathy, Mintz et al.
demonstrated that INTERCEPT-FFP supported hemostasis similar to conventional FFP, with no differences in the
use of blood components, clinical hemostasis, or safety.106
In a Phase III trial, Cerus evaluated the safety and effectiveness of INTERCEPT-FFP compared to standard FFP in
a small group of patients with thrombotic thrombocytopenic purpura (TTP). Remission was achieved in 14 of
17 (82%) patients receiving INTERCEPT-FFP, and 16 of 18
(89%) patients receiving standard FFP. Time to remission,
relapse rates, time to relapse, total volume, and number of
FFP units exchanged were not significantly different
between both groups. No antibodies to amotosalen were
detected.107 A hemovigilance program similar to that
established for INTERCEPT platelets is ongoing to document and monitor the safety of INTERCEPT plasma
transfusion.108
Methylene blue processes similar to MacoPharma
Theraflex MB have been used in Europe for over 10 years,
with over 4 million units transfused in various clinical settings. However, there have been no large controlled, randomized clinical trials comparing MB-FFP to standard
FFP. Most patient studies have been small and/or used
laboratory rather than clinical endpoints.
CaridianBCT Biotechnologies has evaluated their
Mirasol-treated plasma in several in vitro studies and it
has been demonstrated to meet the 14th Edition, Council
of Europe Guidelines for protein content for standard
FFP (Raymond Goodrich, pers. comm., 2009).
22S TRANSFUSION
Plasma clinical experience
Over 6 million units of Octapharma octaplas have
been transfused, and it has been accepted as therapeutically equivalent to standard FFP. No postmarketing
hemovigilance trials have been published.
MacoPharma Theraflex MB has been registered or is
in routine use in 20 countries, including Germany,
Switzerland, Spain, Greece, Italy, France, Belgium, and
the UK. In spite of laboratory observations demonstrating some loss of coagulation factors, clinical reports have
been generally satisfactory. Postmarketing hemovigilance
studies from Greece and Spain evaluating 8500 units and
88,000 units, respectively, have been reported, with no
adverse events associated with MB-FFP and satisfactory
clinical outcomes.109,110 Castrillo et al.111 also reported a
5-year experience with MB plasma with no adverse reactions observed. MB-FFP in TTP patients has been evaluated in several small studies, comparing MB-FFP to
untreated FFP and its effectiveness is a subject of debate.
Two studies in Spain, one retrospective and one prospective, demonstrated that there was a lower remission rate
and higher volume of FFP required for treating TTP when
using MB-FFP compared to control (untreated) FFP.112,113
It has been hypothesized that this is due to decreased
levels of ADAMTS13, the enzyme generally thought
to be deficient or inhibited in these patients;
however, MB treatment does not affect the activity of
ADAMS13.114,115
Limitations of pathogen reduction
Agents with intrinsic resistance to PR processes include
prions, some nonenveloped viruses such as HAV, and bacterial spores. Furthermore, extraordinarily high-titer
viruses like B19V or HBV may not be inactivated below an
infectious dose. Therefore, surveillance for the emergence
of new agents will remain critical even after the introduction of PR.
Since blood components currently carry very low
risks of infectious agent transmission, any manipulation
to further mitigate known risks will be difficult to justify
if it introduces any material new risk to transfusion
recipients. Critical questions have been raised about
301
short- and long-term safety of PR systems to transfusion
recipients, to blood center and hospital staff who may
be exposed to them, and to the environment in which
they are manufactured, used, and in which they are
disposed.
Potential toxicologic effects of the candidate PR
systems have been examined in many dimensions. These
include acute, subacute, and chronic toxicity, blood component incompatibility, genotoxicity, carcinogenicity, and
impact on reproduction and development.
In general, the residual levels of active PR ingredients
in fully processed blood components are below the limit
of detection in available direct toxicity assays due to
robust removal steps included in the processes. Also,
water-soluble molecules are rapidly excreted with no
accumulation in fat. There are, however, diverse reaction
products derived from the active agents and establishing
large safety margins for these products is difficult due to
dose per volume constraints (John Chapman, pers.
comm., 2009).
Compatibility with cellular elements and plasma proteins is critical. Neoantigenicity with sensitization to
treated components is one aspect. Another is the impact
on the recovery, survival, and function of blood elements.
It is important to understand whether or not PR will
increase transfusion requirements related to loss of therapeutic product, or if processing disturbs the delicate
balance in physiologic pathways like coagulation.
All PR methods in development for cellular blood
products rely on interactions with microbial nucleic
acids, raising the specter of carcinogenicity and mutagenicity, and of adverse impacts on reproduction and
development. The study of these potential effects is very
difficult, expensive, and time-consuming. While in vitro
and animal studies can be completed with reasonable
speed and economy and are reassuring when negative,
the clinical events of interest are expected to be quite
rare and may have very long latent periods until recognition. This raises barriers on the path toward regulatory
approvals. Special, more vulnerable populations may
need additional focus in clinical studies. These include
pregnant and fertile women, the fetus, newborns, and
growing children, those requiring massive acute transfusions, and patients who receive chronic transfusion
support. There needs to be a level of comfort that those
with impaired kidney and liver function are not at
elevated risk of adverse events.
Although the companies have been authorized to
market their products in many countries (Table 9), adoption and routine use of these technologies has been
relatively limited. Blood centers, hospitals, and transfusion services will continually evaluate the cost versus
benefit of each of these technologies; at the present
time, the most efficient and effective process(es) is
unknown.
PATHOGEN REDUCTION AND ITS
POTENTIAL FOR EID AGENTS
The potential utility of PR can be appreciated by reviewing the list of 16 agents assigned red, orange, or yellow
priority in this exercise. The data cited, either for specific
agents or relevant models, suggest that only the vCJD
prion of the red agents, and the CWD prion, human parvovirus B19, and HAV among the others, will likely
escape inactivation from clinically relevant titers in
platelets and plasma with application of the systems
being brought forward. The data available for RBCs are
not adequate to instill great confidence at this point, but
early data certainly support concentrated research
efforts. While some see the absence of a single process
applicable to all components as a barrier to the use of
PR, just the prospect of controlling bacterial contamination of platelets, the most common serious infection
associated with contemporary blood transfusion in the
developed world, is an example of its potential power.
Pathogen reduction would be a useful intervention to
reduce transfusion transmission of the agents responsible for babesiosis, Chagas disease, and malaria. Babesiosis from RBC transfusion is widely understood to be
more prevalent than published reports have suggested,
and antibody to T. cruzi testing has been adopted nearly
universally in the US while emerging evidence demonstrates that T. cruzi transmission may be less common
than had been anticipated when decisions were made to
pursue donor testing. Deferral for minimal risk of
transfusion-transmitted malaria remains a source of
serious donor loss. A rational approach to testing for
these three parasites may be selective screening strategies, but less than universal screening is a strong disincentive for test builders to bring a donor screening assay
through the rigorous and costly regulatory approval
process; thus, market forces may prevent or delay test
development. Potentially, PR bypasses this “one agentone test” approach while protecting both recipients and
the donor supply. Similarly, if the next agent to emerge as
a serious threat is an enveloped virus like HBV, HIV, HCV,
and WNV, it is probable that the approaches in development will be robust, prospectively obviating the need for
deferrals and testing. It is also anticipated that a thorough review of the currently used donor screening questions and tests will be required to assess their continued
need once robust pathogen reduction methods are available. Similarly, well-controlled postmarketing studies will
be needed to determine if adverse outcomes occur as a
result of widespread use of PR and the impact of PR on
transfusion-transmitted disease.
CONFLICT OF INTEREST
Peyton S. Metzel is an employee of Fenwal, Inc. Fenwal,
Inc. manufactures InterSol platelet additive solution and
302
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Platelets
Mirasol
Phase I Completed
Completed
INTERCEPT
Phase III Completed
Completed
CE
Phase I
Theraflex UV
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CE
Completed
FFP
Theraflex MB
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MAA
Octaplas
Phase III Completed
Completed
* Council of Europe approval or Marketing Authorization Approval. A CE Mark alone is not adequate regulatory authority to market a device in many countries in Europe including the UK,
France, and Germany.
† SD-plasma available, similar to Octaplas.
Regulating agency or development status
Development Status, US
Development Status, Europe
US FDA
Canadian MOH
CE Mark or MAA*
For sale In:
• Argentina
• Australia
• Austria
• Belgium
• Brazil
• Canada
• Croatia
• Czech Republic
• Finland
• France
• Germany
• Greece
• Gulf Central Committee
• Hungary
• Iceland
• Ireland
• Italy
• Kazakhstan
• Kuwait
• Luxembourg
• Malaysia
• Mexico
• Netherlands
• New Zealand
• Norway
• Oman
• Poland
• Portugal
• Romania
• Russia
• Saudi Arabia
• Singapore
• Slovakia
• Slovenia
• Spain
• Sweden
• Switzerland
• Thailand
• Turkey
• UAE
• UK
• Vietnam
• Yemen
TABLE 9. Development/regulatory status
STRAMER ET AL.
19. Lefrère JJ, Hewitt P. From mad cows to sensible blood
is the contract disposables manufacturer for Cerus Corporation INTERCEPT platelets and plasma. No other conflicts of interest were declared.
transfusion: the risk of prion transmission by labile blood
components in the United Kingdom and in France. Transfusion 2008;49:797-812.
20. Reesink HW, Engelfriet CP, Hyland CA, Coghlan P, Tait B,
Wsolak M, Keller AJ, Henn G, Mayr WR, Thomas I, Osselaer JC, Lambermont M, Beaten M, Wendel S, Qiu Y,
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Sang 2006;91:177.
Sang 2007;93(Suppl 1):167.
104. Hambleton J, Wages D, Radu-Radulescu L, Adams M,
111. Castrillo A, Eiras A, Cid J, Castro A, Adelantado M, Abalo
Mackenzie M, Shafer S, Lee M, Smyers J, Wiesehahn G,
Corash L. Pharmacokinetic study of FFP photochemically
M, Areal C, Flores J, Cabrera J. Quality of methylene blue
plasma over the last five years. Vox Sang 2006;91:182.
treated with amotosalen (S-59) and UV light compared to
FFP in healthy volunteers anticoagulated with warfarin.
112. de la Rubia J, Arriaga F, Linares D, Larrea L, Carpio N,
Marty ML, Sanz MA. Role of methylene blue-treated or
fresh-frozen plasma in the response to plasma exchange
105. de Alarcon P, Benjamin R, Dugdale M, Kessler C, Shopnick R, Smith P, Abshire T, Hambleton J, Matthew P, Ortiz
in patients with thrombotic thrombocytopenic purpura.
Br J Haematol 2001;114:721-23.
I, Cohen A, Konkle BA, Streiff M, Lee M, Wages D, Corash
L. Fresh frozen plasma prepared with amotosalen HC1
113. Alvarez-Larrán A, Del Río J, Ramírez C, Albo C, Peña F,
Campos A, Cid J, Muncunill J, Sastre J-L, Sanz C, Pereira
(S-59) photochemical pathogen inactivation: transfusion
of patients with congenital coagulation deficiencies.
Transfusion 2005;45:1362-72.
A. Methylene blue-photoinactivated plasma vs. freshfrozen plasma as replacement fluid for plasma exchange
in thrombotic thrombocytopenic purpura. Vox Sang 2004;
106. Mintz PD, Bass NM, Petz LD, Steadman R, Streiff M,
McCullough J, Burks S, Wages D, Van Doren S, Corash L.
86:246-51.
114. Yarranton H, Lawrie AS, Purdy G, Mackie IJ, Machin SJ.
Photochemically treated fresh frozen plasma for transfusion of patients with acquired coagulopathy of liver
Comparison of von Willebrand factor antigen, von
Willebrand factor-cleaving protease and protein S in
disease. Blood 2006;107:3753-60.
107. Mintz PD, Neff A, MacKenzie M, Goodnough LT, Hillyer
C, Kessler C, McCrae K, Menitove JE, Skikne BS, Damon L,
blood components used for the treatment of thrombotic
thrombocytopenic purpura. Transfus Med 2004;14:
39-44.
Lopez-Plaza I, Rouault C, Crookston KP, Benjamin RJ,
George J, Lin JS, Corash L, Conlan MG. A randomized,
115. del Río-Garma J, Pereira A, Arroyo JL, Mateo J, AlvarezLarrán A, Martínez C, Muncunill J, Barbolla L; Spanish
controlled Phase III trial of therapeutic plasma exchange
with fresh-frozen plasma (FFP) prepared with amotosalen
and ultraviolet A light compared to untreated FFP in
thrombotic thrombocytopenic purpura. Transfusion 2006;
46:1693-704.
Group of Apheresis (Grupo Español de Aféresis GEA).
ADAMTS-13 activity and von Willebrand factor levels in
methylene blue photo-inactivated plasma and processed
by either the Springe method or an “in house” system.
Vox Sang 2008;95:101-5.
308
TRANSFUSION 29S
August 200545813351341Original Article
Blackwell Science, LtdOxford, UKTRFTransfusion0041-11322005 American Association of Blood Banks
B2 AND UV LIGHT FOR PRTAUBUCHON ET AL.
BLOOD COMPONENTS
文献20
Efficacy of apheresis platelets treated with riboflavin and
ultraviolet light for pathogen reduction
James P. AuBuchon, Louise Herschel, Jill Roger, Harry Taylor, Pamela Whitley, Junzhi Li, Rick Edrich,
and Raymond P. Goodrich
A
dvances in donor screening and testing have
reduced the risks of transmission of human
immunodeficiency virus and hepatitis C virus
by more than 10,000-fold in the past two
decades.1 Diversion and detection methods have been
introduced to reduce the risks of bacterial contamination
in platelets (PLTs).2 Despite these efforts, risks of viral and
bacterial contamination remain. Furthermore, various
parasitic risks have yet to be addressed successfully,
including those of Chagas disease, and the evolution or
introduction of new viruses into the blood supply may
cause significant morbidity and mortality before their recognition can lead to the development and implementation of effective testing.3 Therefore, important health
benefits may accrue with the introduction of pathogen
reduction technologies (PRTs) in blood components.
Many recipients of PLT transfusions are especially
vulnerable to the presence of contaminating pathogens.
Their immunosuppressed and/or neutropenic states may
blunt effective response to a pathogen. Although some
recipients of PLT transfusions may succumb to their
BACKGROUND: Pathogen reduction technologies for
platelet (PLT) components offer a means to address
continued viral transmission risks and imperfect bacterial
detection systems. The efficacy of apheresis PLTs treated
with riboflavin (vitamin B2) plus ultraviolet (UV) light
(Mirasol, Navigant Biotechnologies) was investigated in a
single-blind, crossover study in comparison to untreated
PLTs.
STUDY DESIGN AND METHODS: Normal subjects
(n = 24) donated PLTs by apheresis on two occasions at
least 2 weeks apart. Units were randomized to control or
test arms, the latter receiving the addition of 28 mL of
500 mmol per L B2 and exposure to 6.2 J per mL UV light.
PLTs were stored for 5 days with biochemical and
hematologic analyses performed before and after
illumination on Day 0 and at the end of storage. An aliquot
of each unit was radiolabeled and returned to determine
recovery and survival.
RESULTS: The PLT content of treated units was
maintained from Day 0 (4.1 ¥ 1011 ± 0.4 ¥ 1011) to Day 5
(4.0 ¥ 1011 ± 0.4 ¥ 1011). Treatment with B2 plus UV light
was associated with an increase in lactate production with
concomitant increases in glucose consumption. pH
(control, 7.38 ± 0.07; test, 7.02 ± 0.10) was well
maintained throughout storage. Recovery of treated PLTs
(50.0 ± 18.9%) was reduced from that of control PLTs
(66.5 ± 13.4%); survival was similarly shortened
(104 ± 26 hr vs. 142 ± 26 h; p < 0.001).
CONCLUSIONS: PLTs treated with B2 plus UV light
demonstrate some alterations in in vitro measures but
retain in vitro and in vivo capabilities similar to pathogenreduced and licensed PLT components that have been
shown to have useful clinical applicability. The recovery,
survival, and metabolic properties of Mirasol PLTs should
provide sufficient hemostatic support in thrombocytopenia to justify patient clinical trials.
ABBREVIATIONS: B2 = riboflavin (vitamin B2); PRT(s) =
pathogen reduction technology (-ies).
From the Dartmouth-Hitchcock Medical Center, Lebanon, New
Hampshire; the Eastern Virginia Medical School and American
Red Cross Blood Services, Norfolk, Virginia; and Navigant
Biotechnologies, Inc., Lakewood, Colorado.
Address reprint requests to: Ray Goodrich, PhD, Navigant
Biotechnologies, 10811 West Collins Avenue, Lake Wood, CO
80215-4498.
This study was conducted under contract to Navigant
BioTechnologies, Lakewood, CO. The apheresis equipment was
provided by Gambro BCT, Lakewood, CO. J.P.A. has served as a
consultant to Navigant Biotechnologies, and J.P.A. and H.T. have
received research support from both companies.
Received for publication December 7, 2004; revision
received January 24, 2005, and accepted January 25, 2005.
doi: 10.1111/j.1537-2995.2005.00202.x
TRANSFUSION 2005;45:1335-1341.
309
Volume 45, August 2005
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1335
AUBUCHON ET AL.
to inadvertent duplicate illumination of the PLT component, intercurrent illness (influenza), venipuncture
difficulties, psychiatric hospitalization (owing to an undisclosed existing condition), and incomplete sample collection; a total of 24 paired data sets (17 men, 7 women) were
thus generated.
Subjects donated 1 unit of leukoreduced apheresis
PLTs on an automated blood collection system (Trima
Accel, Gambro BCT, Lakewood, CO) on two occasions at
least 2 weeks apart with a target of 1640 ¥ 103 PLTs per mL
in 270 mL of plasma–acid citrate dextrose (ACD)-A. PLTs
were placed in ELP bags composed of polyvinyl chloride
plasticized with N-butyryl tri-n-hexyl citrate.24 Units were
held undisturbed immediately after collection for 2 to
3 hours to allow dissociation of any PLT aggregates. The
PLT content of each unit was verified to be in the target
range of 1400 ¥ 103 to 1880 ¥ 103 PLTs per mL 2 to 4 hours
after collection; if necessary, the concentration was
reduced to within the allowed range with some of the
150 mL of plasma that had been collected at the time of
the plateletpheresis. The protocol allowed for a repeat collection if any unit failed to meet the content criterion or
other problems developed that precluded successful analysis and return of the unit.
Units were designated to undergo treatment with B2
plus UV light (Mirasol, Navigant Biotechnologies, Lakewood, CO; treated arm) or not (control arm) according to
a statistical package randomization scheme provided by
the sponsor. (If subjects were withdrawn from the study,
they were replaced by subjects who assumed their place
in the randomization table.) At 2 to 4 hours after collection, a volume of 250 ± 5 mL (256 g) of PLTs was gravimetrically transferred from the ELP collection bags to the PLT
illumination-storage bag to be either Mirasol-treated or
stored without further processing (control). B2 solution
(500 mmol/L, 28 ± 1 mL) was added to the test unit through
a sterile barrier filter with a syringe. After thorough mixing, a 3- to 5-mL sample was withdrawn for in vitro assessments. The unit was then secured in a prototype
illumination device and exposed to 5.0 J per cm2 of UV
light (corresponding to 6.2 J/mL delivered to the PLT unit),
a process that took 8 to 10 minutes in the temperaturecontrolled environment of the illuminator (illuminating
wavelengths, 265-370 nm). After the process, a 3- to 5-mL
postillumination sample was obtained from the test unit
for in vitro assessments. One 3- to 5-mL sample was
obtained from the control product for in vitro assessments. The units (control and treated) were stored in the
same PLT illumination-storage bag under normal blood
banking conditions of 22 ± 2∞C with horizontal agitation
for 5 days. On Day 3 or Day 4, a 3- to 5-mL sample was
taken for bacterial culture to document sterility.25 At the
end of the 5-day storage period, a 3- to 5-mL sample was
withdrawn from the products for in vitro assessments and
radiolabeling. (All sampling was conducted with a sterile
underlying disorder, the success of more effective treatments for malignancies over the past several decades is
eliminated if the patient dies because of a transfusiontransmitted infection. Thus, the means to reduce the
infectivity and pathogenic potential of contaminating
organisms in PLT components remains an important goal.
The success of a proposed PRT may be judged in
terms of safety and efficacy.4 The treatment must not, in
itself, create more risk than it is removing, and it must
reduce the presence of viable or infective pathogens to a
clinically useful degree. Similarly, the transfusible component must be well tolerated and retain a sufficient proportion of its efficacy (viability and thrombogenicity in
the case of PLTs) to achieve the clinical aims of the
transfusion.
Riboflavin (vitamin B2) has been shown to interact
with genomic material and irreversibly, covalently combine with RNA and DNA to preclude transcription and
translation of pathogens’ genetic material on exposure
to ultraviolet (UV) light and/or to cause breakage of the
chromosomal strand.5-8 The process has been shown to be
effective against viruses, bacteria, and parasites with sufficient reductions in infectivity (generally ≥ 5 log) to provide a useful safeguard for transfusion recipients.9-14 The
low toxicologic concern regarding use of a vitamin to
achieve this task has been bolstered by embryofetal,
subchronic, and high-dose toxicology studies; pharmacokinetic analysis; neoantigenicity, cytotoxicity, and
hemocompatibility experiments; chromosome aberration
and Ames tests; and a mouse micronucleus mutation
assay, all of which failed to demonstrate results suggestive
of human toxicity.15-20 In vitro analyses of treated, plasmasuspended PLTs indicated good retention of biochemical
and functional variables through 5 days of storage following treatment.21,22
To move investigation of the effect of treating PLTs
with B2 and UV light to the next stage, we conducted a
crossover study in blinded normal subjects to determine
the in vivo efficacy of treated versus control (untreated)
PLTs after 5 days of storage via autologous recovery and
survival studies of radiolabeled PLTs.
MATERIALS AND METHODS
The study received the approval of local institutional
review boards at each participating location and was conducted under an investigational device exemption from
the FDA. Twenty-nine normal, healthy adult subjects (20
men and 9 women, all without reproductive potential)
who met all FDA and AABB23 criteria for PLT donation
pertaining to the health of the donor were recruited into
the study at two locations (15 at one site; 14 at the other)
without regard to previously obtained recovery and survival results. Written informed consent was obtained from
subjects. Five subjects did not complete the study owing
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B2 AND UV LIGHT FOR PRT
positive swirl (inhomogeneity readily visible throughout
the entire bag, 3), intermediate swirl (some inhomogeneity visible, 1), and negative swirl (turbid, 0).
Results are expressed as means ± 1 standard deviation
(SD). Statistical analysis was conducted comparing control versus treated units with a t test statistic with a p value
of less than 0.05 used to reject the null hypothesis of
no difference. Paired analyses were applied wherever
appropriate.
connecting device. All unit handling and processing was
performed at each test site.)
PLTs were labeled with standard techniques before
return into the original donor with standard techniques.26,27 A 10-mL aliquot was removed from the unit,
and the PLTs were concentrated by centrifugation.
Approximately 100 mCi of the radiolabel (111In-oxine,
Medi-Physics, Arlington Heights, IL) were added to a suspension of the PLTs for a 20-minute room temperature
incubation. The PLTs were then washed with a mixture of
plasma and ACD-saline. By use of a dose calibrator, 10 to
20 mCi of the labeled PLTs was taken up into a syringe for
return into the subject. (The remainder was used for standards and quality control procedures.) Samples were
taken from the contralateral arm within the first 3 hours
after return as well as daily for 1 week (for determination
of the proportion of injected label recoverable) and again
on Day 10 (to allow for correction of activity associated
with red blood cells). 111In emissions were counted at 176
to 190 keV. Recovery was calculated from the injected
radioactivity (corrected for preinjection radiolabel
elution27), a formulaic projection of the subject’s blood
volume,27 and back-extrapolation to the time of injection
with the multiple-hit model on a standardized computer
program28,29 and verified by a separate calculation with
a validated computer program (SAS Institute, Inc., Cary,
NC). Survival was recorded as the mean residual life span
determined by the multiple-hit model.
In vitro analyses were conducted on Day 0 (before
treatment in the test units) and on the last day of storage.
PLT concentration was determined from a CBC assessment with a hematology analyzer (Advia 120, Bayer Diagnostics, Tarrytown, NY; or 9110-plus Baker Instruments,
Allentown, PA). For this purpose, a sample was transferred
to an ethylenediaminetetraacetate tube, which was
rotated at 22 ± 2∞C until the sample was analyzed. The
lactate and glucose concentrations were determined on
an automated lactate-glucose analyzer (either the ABL705, Radiometer-America, Westlake, OH; or the Hitachi
917, Hitachi High-Technologies, Tokyo, Japan). The rates
of lactate production and glucose consumption during
the 5 days of storage were calculated with a concentration
normalized to the initial PLT concentration. Unit pH was
analyzed on an automated blood gas analyzer (ABL-705,
Radiometer-America; and Model 855, Bayer Diagnostics)
at 37∞C and converted to pH22∞C algebraically. O2 and CO2
partial pressures were analyzed on an automated blood
gas analyzer (ABL-705, Radiometer-America; or Model 248
or 855, Bayer Diagnostics). Expression of CD62 or GMP140 (P-selectin) was determined by flow cytometry within
14 days after fixation in 1 percent paraformaldehyde per
the method recommended by the Biomedical Excellence
for Safer Transfusion (BEST)30 based on fluorescence
intensity. PLT morphology was characterized with a visual
swirl scoring technique with a three-tiered scoring system:
RESULTS
All subjects participating in the study had no adverse
events related to their participation. All PLT returns were
well tolerated. All unit cultures were negative. Ten repeat
apheresis procedures were performed in nine subjects.
Five repeat apheresis collections were required in the
experimental arm and four were required in the control
arm owing to factors such as intercurrent illness precluding return and handling of units in manners not consistent with the study design. One apheresis procedure had
to be aborted because of infiltration.
The PLT concentrations were 10 percent lower in the
treated units reflecting the dilution caused by the addition
of the B2 but total PLT counts remained unchanged
(Fig. 1). In addition, there was no difference in PLT count
between before treatment to the end of storage in the
treated units.
Treated units demonstrated accelerated glycolysis
with lower glucose concentration at the end of storage,
higher lactate concentration, and increased rates of consumption of glucose and production of lactate (Figs. 2A
and 2B). There was, however, residual glucose in all units
at the end of the storage period, and the pH value,
although lower in treated units, remained above 6.8 in
all units (Fig. 2E). Blood gas measurements showed a
decrease in pO2 and pCO2 during the storage period
Fig. 1. PLT concentration. Results of testing before and after
treatment (Day 0) and at end of storage (Day 5). () Mean of
control units; ( ) treated units. Vertical bars represent 1 SD.
311
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AUBUCHON ET AL.
Fig. 2. Results of testing before and after treatment (Day 0) and at end of storage (Day
5). () Mean of control units; ( ) treated units. Vertical bars represent 1 SD. (A) Glucose
concentration and consumption rate. (B) Lactate concentration and consumption
rate. (C) Oxygen and carbon dioxide partial pressures. (D) P-selectin expression and
swirling. (E) Unit acidity.
1338
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312
(Fig. 2C). The Day 0 and Day 5 results
were not different between control and
treated units’ pO2; pCO2 was significantly lower in test units before treatment but higher at the end of storage.
P-selectin expression increased throughout storage in both groups of units and
was higher on Day 5 in test than control
units (Fig. 2D). The assessment of swirling was not different at the beginning
of storage but was lower on Day 5 in
treated units. The differences in in vitro
measures between control and treated
units at the end of the storage period (as
shown in the figures) were all significant
except for pO2 and pCO2.
Radiolabel was taken well by both
test and control PLTs on Day 5 (Table 1).
The recovery of radiolabeled PLTs from
two subjects’ control units exceeded
biologic possibility (i.e., recovery
>100%) for reasons that could not be
determined. These control units’ in vivo
results were excluded from further analysis, but the treated units’ results were
included in (unpaired) analyses. The
mean recovery and survival of treated
PLTs were reduced when compared with
the recovery and survival of the control
units (Table 1).
Possible relationships between in
vitro measures and in vivo PLT recovery
were analyzed by means of linear regression across both types of units (Table 2).
The best correlations with in vivo recovery were seen for the rate of glucose
consumption (p = 0.0000), lactate concentration (p = 0.0000), rate of lactate
production (p = 0.0001), pH (p = 0.0002),
glucose concentration (p = 0.0031), and
P-selectin (p = 0.0078). The in vitro variable that correlated most closely with
survival was Day 5 pH (p = 0.0021). With
stepwise multiple regression, recovery
and survival were each fitted against all
in vitro variables. It should be noted that
there are strong correlations between
many of the in vitro variables themselves, which has a confounding effect
on obtaining a definitive model. In the
case of recovery, the final regression
model obtained by the stepwise regression method contained only the rate of
glucose consumption. In the case of
survival, pH was the only variable main-
UV light treatment as part of PRT appear to have negative
effects on PLT physiology,31 the Mirasol system appears to
have less of an effect on the number of PLTs available
remaining in the unit through the treatment process.
The reductions in recovery and survival can be
assessed in several different ways. Because the laboratories participating in this study use the same radiolabeling
DISCUSSION
procedure and have participated jointly in other studies,
the results of this study can be usefully compared. The
This randomized crossover study in normal subjects docrecoveries and survivals reported here for the control units
umented that treatment of leukoreduced apheresis PLTs
were very similar to those reported in a study on
with B2 plus UV light to effect pathogen reduction causes
(untreated) leukoreduced PLTs after 5 days of storage
a reduction in radiolabeled autologous recovery and sur(recovery, 63.0 ± 11.2%; survival, 161 ± 38 hr)32 giving convival but retention of sufficient in vivo efficacy to predict
clinical utility. The in vitro analyses indicated that the
fidence that the variability introduced into such analyses
treated PLTs were undergoing glycolysis at an accelerated
over time and over different subjects is minimal when a
rate compared to untreated controls. This metabolic substandardized protocol is used. Furthermore, the recovery
strate, however, was not exhausted during a 5-day storage
of untreated PLTs stored for 7 days (53.9 ± 13.8%), which
period, and the acidity of the treated units, although
was adequate for FDA acceptance of approval for 7-day
greater than for control units, did not exceed the capabilstorage on Trima PLTs, was very close to that observed
ities of the PLTs to maintain functional metabolism. One
with treated PLTs stored for 5 days in this trial
may speculate that a “hypermetabolic state” may be
(50.0 ± 18.9%). (The survival of treated PLTs at 5 days of
induced by the treatment, but the magnitude of this does
storage, 104 ± 26 hr, was shorter than that of untreated
not preclude continued viability of the cells. The effect of
PLTs at 7 days, 134 ± 45 hr, p < 0.05.)
the treatment process was also seen through increased PBoth of these proposed modifications to PLT systems
selectin expression and reduced swirling at the end of the
(extended storage to reduce outdating and pathogen
storage period in comparison with untreated control
reduction treatment to reduce infectious risks) offer useful
units. Importantly, the PLT concentrations, although lowadvantages but obviously carry with them certain downered in the test units by the addition of 28 mL of solution
sides, including reduced efficacy. A trade-off is inevitable
containing the B2, were well maintained through treatin such situations, and value judgments will need to be
ment and storage. Thus, although all systems that utilize
made to determine whether a reduction of in vivo efficacy
is mitigated by reducing the risk of certain pathogens. It
is, however, encouraging to note that
Mirasol PLTs at 5 days of storage appear
TABLE 1. Results of radiolabeling studies*
similar to untreated PLTs at 7 days of
Control
Treated
p value
storage, an approach that has been
Radiolabel uptake efficiency (%)
64.2 ± 17.2
59.5 ± 21.2
>0.05
shown to have clinical utility.25 Because
Recovery (%)
66.5 ± 13.4
50.0 ± 18.9
<0.05
comparison of a new approach to PLT
Survival (multiple hit, hr)
142 ± 26
104 ± 26
<0.05
handling (such as PRT) to a previously
* Results of Day 5 radiolabeling with 111In and autologous return. Results shown are 22
licensed method may lead to “creeping
observations from the control cycle and 24 from the treated cycle.
inferiority” via repetitive comparisons,
tained in the model. When these analyses were repeated
by also including treatment (control vs. treated) in the
model, only the rate of glucose consumption remained as
a significant predictor of recovery. In the model for survival, only treatment was significant.
TABLE 2. Correlation via linear fit of Day 5 in vitro measures with recovery and survival
Measure
PLT concentration
Glucose concentration
Glucose consumption rate
Lactate concentration
Lactate production rate
pH22∞C
pO2
pCO2
P-selectin expression
Swirling
Correlation with recovery
Correlation
coefficient (r)
F value
0.3235
5.1422
0.4269
9.8054
-0.5981
24.5106
-0.5836
22.7236
-0.5608
20.1895
0.5263
16.8536
-0.2061
1.9527
-0.0829
0.3047
-0.3875
7.7734
0.1275
0.7270
313
p value
0.0283
0.0031
0.0000
0.0000
0.0001
0.0002
0.1693
0.5838
0.0078
0.3985
Correlation with survival
Correlation
coefficient (r)
F value
0.1651
1.2327
0.3514
6.1989
-0.3505
6.1619
-0.3564
6.4036
-0.3652
6.7707
0.4426
10.7206
-0.0725
0.2325
-0.0272
0.0325
-0.1767
1.4179
0.2704
3.4710
p value
0.2729
0.0166
0.0169
0.0150
0.0126
0.0021
0.6320
0.8578
0.2401
0.0691
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AUBUCHON ET AL.
an alternative approach to evaluating in vivo efficacy has
been suggested wherein the recovery and survival of PLTs
handled in the new system are compared to fresh PLTs
from the same subject.33 This proposal, which requires the
“test” PLTs to demonstrate at least two-thirds the recovery
and one-half the survival of fresh PLTs in the same subject,
has been successfully validated as providing a realistic
and reproducible benchmark.34,35 The current study was
designed before the proposal for comparison with fresh
PLTs. Use of a standardized protocol for preparation of the
fresh PLTs yields recoveries in the range of 60 to 65 percent
and survivals approximating 200 hours.26,36 Applying the
target proportions, one would expect that acceptable performance would fall into the range of 40 to 44 percent
recovery and approximately 100 hours of survival. The
Mirasol-treated PLTs prepared in this study would thus
appear to meet the new standard, but a direct comparison
with noninferiority statistical comparison would, of
course, be needed to verify this.37
A similar study has recently been published with
amotosalen and UV light to effect PRT in PLTs.38 A direct
comparison of in vivo results would suggest that Mirasoltreated PLTs offered improved recovery and survival,
although technical differences cannot be excluded as the
source of this.
This study has documented the effects of applying a
PRT system for PLTs that has a low potential for toxicity.
The system yields PLTs that clearly show the effect of the
treatment, in terms of altered biochemical analyses and
reduced recovery and survival, but that retain sufficient
viability and functionality to predict their clinical utility.
This study therefore would suggest that clinical trials to
investigate the effectiveness of Mirasol PLTs in thrombocytopenic patients are warranted.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
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2001;20:404.
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the genotoxic potential of riboflavin and lumiflavin. A. Effect
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Hardwick CC, Herivel TR, Hernandez SC, Ruane PH,
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TRANSFUSION
1341
文献21
Mutation Research, 298 (1992) 9-16
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1218/92/$05.00
9
MUTGEN 01821
Assessment of the genotoxic potential of riboflavin and lumiflavin
A. Effect of metabolic enzymes
Hema Kale, P. Harikumar, P.M. Nair and M.S. Netrawali
Food Technology and Enzyme Engineering DiL'ision, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India
(Received 28 January 1992)
(Revision received 13 May 1992)
(Accepted 20 May 1992)
Keywords: Riboflavin; Lumiflavin; Genotoxicity; $9; Caecal cell-free extract
Summary
The mutagenic potential of riboflavin and its photodegradation product lumiflavin was evaluated using
the u m u test, SOS chromotest and Ames Salmonella assay. Both riboflavin and lumiflavin by themselves
were found to be non-mutagenic. On treatment with rat liver microsomal enzymes ($9) or caecal cell-free
extract (CCE), lumiflavin acquired mutagenicity, while the status of riboflavin remained unaffected.
Activation of lumiflavin by metabolic enzymes was found to result in an alteration of its spectral
characteristics.
Riboflavin, a vitamin, has found widespread
application in food products both as a nutrient
and as a colouring agent (Counsell et al., 1981).
Unlike many other food colourants it has commanded unquestioned favour from regulatory
agencies all over the world (Horwitt, 1972; FAO,
1981), since earlier toxicological studies have
failed to reveal any deleterious effects (Purchase
et al., 1978; Haveland-Smith, 1981). However,
evidence obtained by many other investigators
has indicated that riboflavin when exposed to
light could produce mutagenic (Griffin et al.,
1981; Bradley and Sharkey, 1977; Pathak and
Carbonare, 1988) as well as cytotoxic effects (Lee,
1969; Misra et al., 1987, 1990) in eukaryotic cells.
Synergistic effects of riboflavin with light have
also been shown to bring about alterations in
DNA and in individual nucleotides in vitro
(Uehara, 1966; Speck et al., 1976; Korycka-Dahl
and Richardson, 1980; Alvi et al., 1984). These
studies clearly indicated the toxic potential of
riboflavin. However, the molecular mechanisms
associated with the interaction of riboflavin or its
photodegradation product lumiflavin with living
cells are poorly understood.
In the present report an attempt has been
made to assess the mutagenic potential of riboflavin as well as its photodegradation product
lumiflavin using three short-term tests, namely
the u m u test, SOS chromotest and Ames/Salmonella assay.
Correspondence: Dr. P. Harikumar, Food Technology and
Enzyme Engineering Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India.
316
10
following day the animals were killed by cervical
dislocation. Livers were removed under sterile
conditions into ice-cold 0.15 M KCI and $9 fraction was prepared according to the method of
Maron and Ames (1983). The tissue was homogenised in 0.15 M KCI using a Potter Elvehjem glass homogeniser with 5 up and down strokes
of a teflon pestle (25% homogenate). The homogenate was then centrifuged at 9000 × g for 10
min and the supernatant ($9 fraction) was decanted and was dispensed in 2-ml aliquots in
sterile plastic tubes which were frozen quickly in
liquid nitrogen and stored at - 8 0 ° C until use.
The $9 mixture was prepared freshly before use
(Ames et al., 1975) by incorporating 4 mM NADP,
8 mM MgCI 2 and 33 mM KCI to a 1 : 10 diluted
fraction. Dilution for SOS chromotest was carried
out with Luria broth instead of a buffer. The
mixture was sterilised using a 0.45-/xm Milliporc
filter. The metabolic activity of $9 was ascertained using the Ames/Salmonella assay (Ames
et al., 1975). After activation benzo[a]pyrene exhibited 900 additional revertants.
Materials and methods
Riboflavin (7,8-dimethyl-10-(D-ribityl) isoalloxazine) was a kind gift from Glindia Ltd. (Bombay,
India). Lumiflavin (7,8,10-trimethyl isoalloxazine),
4-nitroquinoline-N-oxide (4NQO), 9-aminoacridine (9AA), mitomycin C (Mit C), nalidixic acid,
benzo[a]pyrene (Bap), rutin, nicotinamide adenine dinucleotide phosphate (NADP), glucose-6phosphate (G-6-P), L-histidine. HC1, biotin, pnitrophenyl phosphate (PNPP), sodium dodecyl
sulfate (SDS) and O-nitrophenyl-a-D-galactopyranoside ( O N P G ) were purchased from
Sigma Chemical Company (St. Louis, MO, USA),
4-dithio-DL-threitol (DTT) and N-methyl-N'nitro-N-nitrosoguanidine (MNNG) were acquired
from Fluka A G (Germany). Aroclor 1254 was
obtained from Monsanto (St. Louis, MO, USA).
Nutrient broth, tryptone and yeast extract were
acquired from Difco Laboratories (Detroit, MI,
USA). All other chemicals and solvents were of
AR grade, the latter were distilled before use.
The purity of riboflavin and lumiflavin was ascertained by TLC using silica gel with two different
solvent systems, viz. pyridine-glacial acetic acidwater ( 1 0 : 1 : 4 0 , v / v ) and n-butanol-acetic
acid-water (40 : 10 : 50, v/v).
Salmonella typhimurium TA100, TA98 and
TA97a were kindly provided by Prof. Bruce N.
Ames (University of California, Berkeley, CA,
USA). Salmonella typhimurium TA1535/psk 1002
was obtained from Dr. Yoshimitsu Oda (Osaka
Prefectural Institute of Public Health, Nakamachi-1, Japan) while Escherichia coli PQ37 was
acquired from Dr. Maurice Hofnung (Institut
Pasteur, Paris, France). Characteristics of the
strains were confirmed and frozen permanents
prepared according to the published protocols
(Maron and Ames, 1983; Quillardet and Hofnung, 1985). Working stock cultures of Ames
strains were maintained on nutrient agar slants
and those of other strains on Luria agar slants
and were stored at 0-4°C until use.
Caecal cell-free extract
Caecal cell-free extract (CCE) was prepared
following the procedure of Brown and Dietrich
(1979). Male Wistar rats weighing about 200-250
g were killed by cervical dislocation and the caecum was removed. The caecal contents were suspended in cold Krebs-Ringer phosphate buffer,
pH 7, containing 2.5 m g / m l D T T for the
Ames/Salmonella assay or umu test or in Luria
broth for the SOS chromotest. The suspensions
were homogenised in a Potter Elvehjem homogeniser and sonicated for 5 rain (Sonics and
Materials Inc.). The sonicated extract was centrifuged at 13,000 × g for 15 min and the supernatant was filtered through a Millipore filter (0.45
/xm) and stored at - 8 0 ° C until use. CCEactivated rutin showed 200 r e v e r t a n t s / 1 0 0
rag/plate with Salmonella typhimurium TA98.
Mutagenicity assays
Preparation of liL,er $9 fraction
Male Wistar rats weighing 200-250 g were
injected intraperitoneally (500 m g / k g body
weight) with Aroclor 1254 (200 m g / m l DMSO).
On the fourth day, feed was removed and on the
(1) Umu test. The general screening of mutagenicity of riboflavin and lumiflavin was carried
out using the umu test (Oda et al., 1985). The
system consisted of 0-100 /xg/ml of test corn-
317
11
(Maron and Ames, 1983). The incubation mixture
consisting of 0.2 ml of a 16-18-h-old culture of
tester strains, Salmonella typhimurium (TA100,
TA98 and TA97a), 0-100 p.g/ml test compounds
and 0.1 ml PBS, pH 7.4, was incubated at 37°C
for 30 min and centrifuged at 3020 × g for 10
min. The pellet was resuspended in 0.4 ml of 0.1
M PBS, pH 7.4. To this suspension 2 ml molten
soft agar was added, mixed rapidly and spread
immediately on preset minimal agar plates. The
activation potential of the metabolic enzymes on
riboflavin and lumiflavin was assessed by repeating the above assay in the presence of 0.1 ml $9
or CCE.
pounds, 2.5 ml of exponentially growing cells
(OD600 adjusted to 0.25-0.3) and 33 mM phosphate buffer, pH 7.4. The mixture was incubated
at 37°C for 180 min and was centrifuged at 3020
x g for 10 min. The pellet was washed 3 times
with 0.1 M phosphate buffer, pH 7.4, resuspended in 3 ml of the same buffer and OD600 was
determined. 0.2 ml of this suspension was added
to 1.8 ml of Z buffer (60 mM N a z H P O 4, 40 mM
NaHzPO4, 10 mM KC1 and 1 mM MgSO4, pH 7)
followed by 50 /~1 SDS (0.1%) and 10/~1 chloroform. After mixing it thoroughly, 0.2 ml O N P G
solution (4 m g / m l in 0.1 M phosphate buffer, pH
7) was added to the reaction mixture and incubated at 28°C for 20 min. The reaction was
stopped by the addition of 1 ml of 1 M Na2CO 3
and the absorbances at 420 nm and 550 nm were
determined. The activity of /3-galactosidase was
calculated according to Miller (1972). The effect
of metabolic enzymes on riboflavin and lumiflavin
was assessed by incorporating 0.5 ml of $9 or
CCE in the original incubation mixture.
Effect of metabolic enzymes on riboflavin and lumiflavin
Aliquots of riboflavin or lumiflavin (160
/~g/ml) were incubated with 3 ml $9 or CCE at
37°C for 60 min. After incubation lumiflavin was
extracted with 3 ml chloroform, the solvent was
evaporated off under nitrogen, the dried residue
was redissolved in methanol and analysed by
spectral scan and TLC. Riboflavin was extracted
with amyl alcohol and quantitated by spectral
scanning and TLC in the same solvent.
(2) SOS chromotest. The assay was performed using the procedure of Quillardet and
Hofnung (1985) with minor modifications. 0.5 ml
of an 18-h-old culture was diluted to 5 ml with
Luria broth (1% Bacto tryptone, 0.5% yeast extract, 1% sodium chloride and 0.002% ampicillin)
and incubated with shaking at 37°C for 3 h; the
optical density at 600 nm was adjusted to 0.25-0.3
(2 x 10 s cells/ml). The cell suspension was diluted to 10 ml with Luria broth and 0.6 ml of the
diluted cell suspension was incubated with 0-100
/~g/ml of test compounds at 37°C for 120 min
and centrifuged at 3020 x g for 10 min. The pellet was washed 3 times with 0.1 M phosphate
buffer, pH 7.4, resuspended in 0.6 ml of the same
buffer and activities of alkaline phosphatase and
/3-galactosidase were determined. The enzyme activities were calculated according to the formula
of Quillardet and Hofnung (1985). The effect of
metabolic enzymes on riboflavin and lumiflavin
was assessed by incorporating 5 ml of $9 or CCE
to 5 ml of cells in place of Luria broth.
Protein
The protein contents of the $9 fraction and
CCE were assessed using Lowry's method with
bovine serum albumin as the standard (Lowry et
al., 1951).
Results and discussion
Data on the mutagenicity of riboflavin and
lumiflavin evaluated with the umu test, SOS
chromotest and Ames/Salmonella assay are presented in Tables 1, 2 and 3 respectively. It was
observed that riboflavin did not demonstrate any
mutagenic response, before and after exposure to
metabolic enzymes. These results are in agreement with the earlier reports on the non-genotoxicity of riboflavin by Purchase et al. (1978),
Haveland-Smith (1981) and Combes and Haveland-Smith (1981).
In contrast to this, lumiflavin after activation
by $9 and CCE showed significant mutagenicity
in the umu test, SOS chromotest and A m e s /
Ames/Salmonella assay. The liquid preincubation procedure was adopted for determining
the mutagenicity of riboflavin and lumifavin
318
12
TABLE 1
m e d i a t e d activity of lumiflavin to an extent of 1.9,
2.9 a n d 4.8 times was also observed in Salmonella
typhimurium TA100, T A 9 8 a n d T A 9 7 a respectively in the A m e s / S a l m o n e l l a assay (Table 3).
Lumiflavin after exposure to C C E showed m u t a genic r e s p o n s e in strain T A 9 7 a by increasing the
histidine r e v e r t a n t s to 3 times the s p o n t a n e o u s
r e v e r t a n t s while the o t h e r two strains r e m a i n e d
unaffected. To our knowledge this is the first
report on the genotoxic p o t e n t i a l of a phot o d e g r a d a t i o n p r o d u c t of riboflavin. M a n y comp o u n d s such as polyaromatics, aromatic amines,
acridines a n d azo c o m p o u n d s are k n o w n to bring
a b o u t frameshift m u t a t i o n s in the A m e s test with
a n d without m e t a b o l i c activation ( v o n d e r H u d e
et al., 1988). O h t a et al. have shown that the SOS
f u n c t i o n - i n d u c i n g activity of 2 - a m i n o a n t h r a c e n e
increased m a r k e d l y in the p r e s e n c e of $9 mix.
T h e significance of m e t a b o l i c enzymes in b r i n g i n g
a b o u t m u t a t i o n in the A m e s test is also highlighted by Prival a n d Mitchell (1982). A l t h o u g h
the precise m e c h a n i s m u n d e r l y i n g lumiflavin-ind u c e d m u t a g e n i c i t y is not clear at present, the
investigations of K u r a t o m i a n d Kobayashi (1977)
with isolated D N A molecules indicated an interaction of lumiflavin with polynucleotides, especially with poly(G). They have suggested that
these i n t e r a c t i o n s could be ascribed to the possible i n t e r c a l a t i o n s of flavins with the D N A bases.
ASSESSMENT OF GENOTOXIC POTENTIAL WITH THE
UMU TEST
Compound
(/xg/ml)
/3-Galactosidase activity~
(Units/OD60o)
1
0
2
97.00_+4.00 136.50+4.10
3
137.66+1.88
Riboflavin
25
50
100
98.00+1.40 144.13_+3.05 136.13_+4.07
101.10_+1.82 144.06+3.61 142.80+4.38
94.28_+3.30 134.23_+3.65 138.66+5.92
Lumiflavin
25
50
100
142.66_+4.26 153.31_+2.25 183.06+5.92
130.20_+3.93 230.10_+6.68 236.18+2.19
129.33 -+7.05 267.43_+8.09 247.80+ 2.76
The values represent mean + SD of 4 independent experiments.
a Activity of /3-galactosidase is calculated as units A420>(
1000/time.
1, Without $9 or CCE; 2, with $9; 3, with CCE.
=
S a l m o n e l l a assay (Tables 1, 2 a n d 3). Lumiflavin
per se was n o n - m u t a g e n i c in any of the test
systems. However, it showed m u t a g e n i c r e s p o n s e
after activation by m e t a b o l i c enzymes. In the
p r e s e n c e of $9 or CCE, lumiflavin elicited a
2-fold increase in /3-galactosidase activity in the
umu test as well as in the SOS chromotest. $9-
TABLE 2
ASSESSMENT OF GENOTOX1C POTENTIAL WITH THE SOS CHROMOTEST
Compound /3-Galactosidase a units
(/xg/ml)
1
2
3
Alkaline phosphatase ~ units
1
2
3
Ratio
/3-gal/AP
1
2
Induction
factor b
3
1
2
3
0
1.49_+0.55 2.10_+0.52 1.81_+0.27 10.50+1.00 11.00_+3.60 10.90_+1.80 0.14 0.19 0.16 1.00 1.00 1.00
Riboflavin
25
50
100
1.66_+0.572.00_+0.50 2.10_+0.45 10.40_+0.50 10.30_+4.20 11.20_+2.50 0.15 0.19 0.18 1.00 1.00 1.10
2.10_+0.45 1.80_+0.52 1.80_+0.26 10.80_+1.30 10.10_+4.20 10.70_+3.70 0.19 0.17 0.16 1.30 0.89 1.00
2.20_+0.57 1.75+0.25 1.60_+0.23 10.70_+2.8 10.70+1.40 11.20_+2.40 0.20 0.16 0.14 1.40 0.84 0.87
Lumiflavin
25
1.56_+0.403.67+0.58 3.90_+0.36 11.40_+4.40 10.90_+2.80 10.30_+0.98 0.14 0.33 0.28 1.00 1.73 1.75
50
1.60_+0.364.00+1.00 3.26_+0.25 10.60_+0.65 10.40_+1.60 10.50+0.50 0.15 0.38 0.31 1.00 2.00 1.93
100
1.80_+0.344.20_+0.26 3.60_+0.36 11.30_+2.20 10.40_+1.20 11.00_+0.85 0.15 0.40 0.32 1.00 2.10 2.00
Values represent mean + SD of 4 independent experiments.
a Activities of/3-galactosidase and alkaline phosphatase are calculated as units - A420× 1000/time.
b Induction factor = R at tested concentration/R at concentration zero.
319
320
95.25+32.20
92.33 _+12.61
60.28 + 12.20
Lumiflavin
25
50
100
139.10.+33.20
146.33 _+24.33
163.37 + 26.40
81.00-+22.50
77.12.+19.85
67.40_+23.19
85.66+21.30
71.50+ 0.70
97.60 + 19.90
66.60 + 15.20
78.16-+32.50
72.00.+23.50
87.80-+14.30
68.30+13.50
Values represent mean _+ SD of 6 independent experiments.
73.37+12.80
79.70.+17.10
85.50.+16.80
Riboflavin
25
50
100
104.40.+15.60
28.60.+3.70
20.85 -+4.37
25.50 + 5.80
18.25.+6.60
17.25.+4.10
23.00.+7.30
26.22.+4.10
1
0
TA98
1
3
TA100
(/xg/ml)
2
Number of revertants
Compound
35.10+ 4.50
48.33 + 7.80
63.40 + 12.80
25.40.+ 3.90
21.66.+ 7.90
22.50.+5.0
21.80.+5.30
2
ASSESSMENT OF GENOTOXIC POTENTIAL WITH THE A M E S / S A L M O N E L L A ASSAY
TABLE 3
25.40+3.80
24.00+3.39
22.40+2.30
186.00+20.21
177.10+28.41
172.90-+16.20
101.00_+10.80
111.50+28.40
88.00+_ 5.70
105.30.+12.41
19.50+5.50
23.20+3.80
24.10_+3.90
1
23.00+5.70
TA97a
3
197.80_+ 29.73
488.18.+108.70
583.12-+ 69.90
99.12_+ 12.50
143.60-t- 27.40
127.25+ 15.40
120.10.+31.19
2
280.40+46.70
295.40.+61.80
360.00-+49.50
105.12+19.70
126.80+24.70
106.80+10.22
119.20.+20.80
3
Uo
14
30I
TABLE 4
TLC OF R I B O F L A V I N AND L U M I F L A V I N A F T E R EXP O S U R E TO THE MAMMALIAN METABOLIC ENZYMES $9 OR CCE
2.4
Sample
Rf
Riboflavin
Lumiflavin
0.71
0.63
Rb + $9
Rb + CCE
0.71
0.72
Lm + $9
Lm + CCE
0.62
0.63
i
•
1-8-
~
•
Riboflavin and lumiflavin were separated by TLC using the
solvent system pyridine-glacial acetic acid-water (20:2:80,
v/v).
\m /
0-0
200
Such intercalations can lead to alteration in the
reading frame of DNA culminating in frameshift
mutations (Auerbach, 1976). Our results do indicate a positive mutagenic response (Table 3) in
tester strains TA98 and TA97a which detect
frameshift mutations. Observation with tester
strain TA100 points to yet another mode of D N A
damage by lumiflavin, namely base-pair substitution. Different mechanisms such as replacement
of bases can lead to base-pair substitution
(Stanier, 1976).
The treatment with the metabolic enzymes $9
and CCE did not alter Rf values of riboflavin
(0.72) and lumiflavin (0.63) (Table 4). No spectral
change was observed with riboflavin, before or
after the treatment with metabolic enzymes (Fig.
1). The absorption maximum of lumiflavin at 221
nm, however, was found to shift to 232 nm (Fig.
2) and to 229 nm respectively on exposure to $9
and CCE (Fig. 3).
It is interesting to note that lumiflavin expresses mutagenicity only after its activation by
metabolic enzymes. Enzymes of $9 and CCE are
known to activate a variety of chemicals by epoxidation, oxidation, reduction, hydroxylation, acetylation and by conjugation reactions (Bartsch et al.,
1982). The near identical spectral characteristics
(Fig. 3) and Rf values (Table 4) observed for
riboflavin and lumiflavin before and after the
treatment with metabolic enzymes suggest that
these compounds did not undergo major structural alterations during activation. However, the
,
I
260
,
I~
I
J
I
u
320
380
WAVELENGTH, nm
I
L
440
504
Fig. 1. Effect of $9 and CCE on the spectral characteristics of
riboflavin. I , Riboflavin; z~, riboflavin+S9; ©, riboflavin+
CCE. Details regarding the treatment of riboflavin with $9
and CCE and the extraction of the products are given in
Materials and methods.
2.0
2.z,I
z 1.E
#
1-6
mxf'X\
./~
I ,
,i
jx~,×
•
r
\\
m~(
o
0"8
..X¢
"/
k\
•
•
X
W
1"2
x
0.C
200
CO
nO
u')
m
.,~ 0.8
I
224
1
I
248
272
WAVELENGTH, n m
~I I~I'l296
320
~1'
m i
jlm
O.Z.
~x
200
260
320
380
WAVELENGTH, nm
440
500
Fig. 2. Effect of $9 on the spectral characteristics of lumiflavin, ll, Chloroform extract of lumiflavin; D, standard lumiflavin; x , lumiflavin + $9. Details regarding the treatment of
lumiflavin with $9 and the extraction of the products are given
in Materials and methods. The spectral pattern at 200-320
nm is expanded in the inset.
321
3.2
15
2"4
2"4
16
i
00,8
;
0200
i
I
760
I
WAVELENGTH,nm
320
260
320
380
440
500
WAVELENGTH. nm
Fig. 3. Effect of CCE on the spectral characteristics of lumiflavin, l , Chloroform extract of lumiflavin; t2, standard lumiflavin; e, lumiflavin + CCE. Details regarding the treatment of
lumiflavin with CCE and the extraction of the products are
given in Materials and methods. The spectral pattern at
200-320 nm is expanded in the inset.
minor (10 nm) shift of peak in the case of lumiflavin indicated that the changes may not be
extensive (Figs. 2, 3 and 4). Other modifications
such as tautomerisation or the production of reactive oxygen species which has the potential to
induce mutagenicity may also be involved (Winston and Cederbaum, 1983; Albano et al., 1988).
The foregoing discussion clearly indicated that
lumiflavin, a photodegradation product of riboflavin, can induce mutagenicity. This is particularly significant because many naturally occurring
food products contain riboflavin which has the
potential to produce lumiflavin-like compounds
(Holmstrom, 1964). Findings in our own laboratory have shown that exposure of riboflavin to
sunlight in aqueous model systems as well as in
natural food products like milk can lead to its
conversion to form lumiflavin.
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Misra, R.B., L.P. Srivastava and P.C. Joshi (1990) Phototoxic
effects of riboflavin in Tetrahymena thermophila, Indian J.
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Oda, Y., S.I. Nakamura, I. Oki, T. Kato and H. Shinagawa
(1985) Evaluation of a new system (umu test) for the
detection of environmental mutagens and carcinogens,
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(1984) The SOS function-inducing activity of chemical
mutagens in Escherichia coli, Mutation Res., 131, 101-109.
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Prival, M.J., and V.D. Mitchel (1982) Analysis of a method for
testing azo dyes for mutagenic activity in Salmonella typhimurium in the presense of flavin mononucleotide and
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Further observations on the photooxidation of DNA in the
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Uehara, K., T. Mizoguchi and S. Hosami (1966) Photooxidation of adenine and its nucleotides in the presence of
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323
文献22
Photochemistry and Photobiology, 2004, 80:
609–615
Technical Note
Separation, Identification and Quantification of Riboflavin and its
Photoproducts in Blood Products using High-performance Liquid
Chromatography with Fluorescence Detection: A Method to Support
Pathogen Reduction Technology{
Christopher C. Hardwick, Troy R. Herivel, Shiloh C. Hernandez,
Patrick H. Ruane and Raymond P. Goodrich*
Navigant Biotechnologies Inc., Lakewood, CO
Received 14 April 2004; accepted 20 September 2004
recovery for RB and a 102% recovery for LC. Samples were
centrifuged and diluted 1:50 with 0.9% saline before analysis.
No protein precipitation or extraction was required. A mass
balance of approximately 93.4–94.4% was achieved after
exposure of products to UV light in the intended pathogen
reduction treatment method. The method permitted the
identification of photoproducts in blood that were both
naturally occurring and produced after photolysis of blood
samples treated with the PRT process. The identity of these
photoproducts has been established using HPLC Tandem
Mass Spectrometry (MS/MS) and UV spectroscopic methods
and has been correlated with known metabolites and
catabolites of RB. HPLC with fluorescence detection using
a reverse phase cyano-column allows for accurate separation,
identification and quantification of both RB and LC in blood
products without the need for solvent extraction or protein
precipitation. Additional photoproducts could also be identified and quantified using this method. The presence of these
agents in normal, untreated blood suggests that their presence
in blood is ubiquitous.
ABSTRACT
A medical device using riboflavin (RB) and light is being
developed for the reduction of pathogens in platelet concentrates (MIRASOLÔ pathogen reduction technology [PRT]). A
high-performance liquid chromatography (HPLC) method for
the quantification of RB and its main photoproduct,
lumichrome (LC) in blood components has been developed
and validated. In addition, the same method has been used to
identify and quantify the presence of additional photoproducts–catabolites of RB. Levels of these agents before
and after treatment as well as endogenous levels present in
normal donor blood are reported using this analytical
technique. The method allows for quantitative and qualitative
analysis of RB and LC in blood components using HPLCfluorescence detection, a ZorbaxÒ SB-CN (stable bond cyano)
column and a methanol–water mobile phase. Quantitation and
qualitative analysis of additional photoproducts of RB was
also performed, but the method has not been validated for
these other components. The method described has passed an
8 day validation and has been found to be adequate for its
intended use. The range of the method for RB is 0.016–1.500
lM and for LC is 0.060–1.500 lM. The method detection limit
for RB is 0.0006 lM and for LC is 0.012 lM. The acceptance
criteria for repeatability were met; the relative standard
deviation for RB was 0.64% and for LC was 0.76%. The
acceptance criteria for bias were met with a 97% average
INTRODUCTION
The collection, separation and transfusion of red blood cells,
platelets, whole plasma and fractionated plasma components are
mainstays of our health care system. Each of these elements is
essential for the preservation of life and the treatment of disease.
Despite years of effort, suitable substitutes have yet to be
developed.
In the early 1980s, it became clear to the medical community
that these life-sustaining and essential therapeutic elements were
transmitting life-threatening diseases (1). The primary causative
agents of these diseases were identified as the human
immunodeficiency virus (HIV) and hepatitis C virus. Because
of the delay in development of suitable detection systems, these
agents passed undetected into the blood supply. The fact that
these potentially fatal diseases were transmitted by blood, and the
lack of suitable blood substitutes, posed a tremendous challenge
for members of the medical community. The resulting concerns
{Posted on the website on 21 September 2004
*To whom correspondence should be addressed: Navigant BiotechnologiesÔ, 1215 Quail Street, Lakewood, CO 80215, USA. Fax: 303-2314011; e-mail: [email protected]
Abbreviations: FAD, flavin adenine dinucleotide; FMF, formylmethyl
flavin; FMN, flavin mononucleotide; HIV, human immunodeficiency
virus; 1H NMR, proton nuclear magnetic resonance; HPLC, highperformance liquid chromatography; HPLC-MS/MS, high-performance
liquid chromatography-Tandem Mass Spectrometry 2KF, 29 keto-flavin;
4KF, 49 keto-flavin; LC, lumichrome; PRT, pathogen reduction
technology; RB, riboflavin; TCA, trichloroacetic acid; UV-VIS, UV–
visible.
Ó 2004 American Society for Photobiology 0031-8655/04
$5.00þ0.00
609
324
610
Christopher C. Hardwick et al.
raised by these events triggered a surge of research into methods
to purify, reduce or eliminate infectious agents in the world’s
blood supply.
We are developing a medical device for the reduction of
pathogens in platelet concentrates. The technology uses light and
the photosensitizer riboflavin (RB) (vitamin B2). The extent of our
interest in RB as a pathogen reduction agent for blood stems from
over 70 years of research literature that details vitamin B2’s
chemistry, toxicology and the in vitro and in vivo function in
sensitizing the photochemistry of nucleic acids (2–4). Interest in
the chemistry of this compound was based on its involvement in
metabolic and nutritional functions as well as its behavior in
individuals subjected to phototherapy (5–8). Before this research,
RB had never been evaluated as a possible sensitizer for use in the
ex vivo photochemical treatment of blood products. The presence
of RB in our diets (9–11), the extensive knowledge of its
photochemical properties and its well-behaved toxicological profile
makes it an ideal candidate for a blood additive in this application
(12,13). Previous studies have demonstrated the ability of this
process to inactivate viruses including Human Immunodeficiency
Virus (HIV), Porcine Parvovirus (PPV) and West Nile Virus
(WNV) and bacteria including S. epidermidis and E. coli in platelet
products while maintaining normal cell quality parameters suitable
for transfusion (14).
One of the concerns about using any chemical agent as an
additive to blood components for use in pathogen reduction
technology (PRT) applications arises from the potential introduction of new chemical agents into the blood supply. These chemical
agents may, on their own accord, raise concerns regarding
increased toxicological risks that could, in certain circumstances,
outweigh the risks associated with potential disease transmission of
the blood component. The toxicological profile of RB is very well
known and characterized. However, after exposure of RB to light,
photochemical reactions can lead to the degradation of the
molecule, yielding several photoproducts (15). These photoproducts result primarily from the decomposition of the ribityl
side chain in the parent molecule (16,17). Several of these
intermediates and breakdown products have been isolated and
characterized (16–19).
Exposure of RB in aqueous solution to light leads to rapid
photobleaching, measured at the absorption maximum of 447 nm
(20). At alkaline pH, lumiflavin is a major breakdown product of
RB (20). Under neutral and acidic conditions (the pH at which the
pathogen reduction process is performed), lumichrome (LC) is the
main breakdown product. Several intermediate by-products produced through metabolic or photochemical degradation of RB have
also been identified. These include the 29 keto-flavin (2KF) and 49
keto-flavin (4KF) and formylmethyl flavin (FMF) (16). The
isolation and characterization of these agents has been hindered
by both the low levels present in isolated samples and the low
sensitivity of methods of isolation and quantification available to
the research community for study of these agents.
The ability to identify these agents in blood components as
they naturally occur and the further ability to quantify or
characterize them in samples before and after PRT treatment
would permit direct examination of the potential impact of PRT
on blood component chemistry and toxicology. Such studies,
although not definitive, could be used to examine potential
consequences in this regard in the blood transfusion setting.
Hence, in support of clinical trials and toxicology studies
involving this treatment regimen, we have developed a novel
Table 1.
Solvent gradient program parameters for HPLC method
Time (min)
Methanol (%)
Water (%)
0.0
8.0
16.0
17.0
18.0
19.0
25.0
23.0
27.8
60.0
100.0
100.0
23.0
23.0
77.0
72.2
40.0
0.0
0.0
77.0
77.0
high-performance liquid chromatography (HPLC) method for the
separation, identification and quantification of RB, LC and other
photoproducts of RB in apheresis platelets both before and after
PRT treatment. Typically, the measurement of flavins in blood
components involves trichloroacetic acid (TCA) precipitation of
proteins (21–34). This evolved from the earlier Warburg and
Christian methods and was used with high salt (usually
ammonium sulfate) both to dissociate flavocoenzymes (flavin
mononucleotide [FMN] and flavin adenine dinucleotide [FAD])
from flavoenzymes and more recently to inactivate enzymes that
could hydrolyze the flavocoenzymes or even catabolize the
released RB. Subsequent extraction and concentration of the
flavins was done with phenol or benzyl alcohol.
In this study we describe a sensitive and robust method for the
accurate determination of both RB and LC in blood components
without the need for protein precipitation or extraction. We also
discuss the findings from direct examination of blood components
before and after addition of RB and exposure to light as is proposed
in the blood sterilization treatment protocol. These results are
presented in terms of potential toxicological and medical
implications for this blood safety initiative.
MATERIALS AND METHODS
Chemicals. HPLC grade methanol was purchased from Fisher Scientific
(Pittsburgh, PA). RB and LC were purchased from Sigma–Aldrich (St.
Louis, MO). RB was used without further purification. LC was purified as
described below. Reagent grade water was prepared using a Barnstead EPure water purification unit (Dubuque, IA). Saline (0.9%) was purchased
from B. Braun Medical Inc. (Irvine, CA).
HPLC apparatus and chromatographic conditions. The HPLC system
consisted of an Agilent 1100 equipped with a quaternary pump (model
G1311A), an autosampler (model G1329A), an autosampler thermostat
(model G1329A), a thermostatted column compartment (model
G1316A), a fluorescence detector (model G1314A) and a fraction
collector (model G1364A). The fluorescence detector flow cell volume
was 8 lL. The HPLC column used was a ZorbaxÒ 80Å SB-CN, 4.6 3
250 mm, 5 micron. The precolumn was a ZorbaxÒ 80Å SB-CN 4.6 3
12.5 mm, 5 micron. The nominal backpressure was 96 bar at the
beginning of the run. The fluorescence detector settings were changed
during each run. Initial fluorescence detector settings were maximized for
detection of RB followed by maximum sensitivity for LC. The settings
were: excitation 268 nm, emission 525 nm for the first 14.5 min,
followed by excitation 260 nm, emission 470 nm. The small change in
baseline upon wavelength change did not significantly affect the
integration. A manual injector program was set up using four wash
vials (reagent grade water) to wash the needle between runs and prevent
carryover of RB and LC into the next run. The left- and right-column
temperatures were set to 508C. The autosampler temperature was set to
58C. Flow rate was 1.0 mL/min. The injection volume was 10 lL. Run
time was 25 min.
Reagent grade water and methanol were used as the mobile phase and
a gradient program was set up (Table 1) to separate RB and LC from the
other photoproducts. A six-point calibration curve was used with RB and
LC concentrations ranging from 250 to 1500 nM. Calibration stock
325
Photochemistry and Photobiology, 2004, 80
611
consisted of 50 lM RB and 50 lM LC in 0.9% saline, pH 5.0. All stock
sample concentrations were confirmed by spectrophotometric analysis of
RB and LC determined from extinction coefficients of the purified
compounds. The stock was diluted to the appropriate calibration
concentrations with 0.9% saline. An excellent straight-line fit was obtained
for both RB and LC (r2 0.999 in both cases). The precolumn was
replaced after every 30–60 samples.
HPLC-MS/MS analysis. Photoproducts of RB were isolated in 10 separate
fractions using a fraction collector. Injection volume was maintained at 500
mL. A diode array detector was used during the separation with settings of
stored range from 190 to 500 nm, peak width ,0.05 min, slit 4 nm.
The following table contains the fraction collection start and stop times:
Fraction
number
Start time
(min)
End time
(min)
Duration
(min)
1
2
3
4
5
6
7
8
9
10
3.9
4.4
5.2
5.6
6.1
6.6
8.9
9.7
11.6
13.2
4.3
4.8
5.5
6.0
6.4
7.0
9.3
10.0
12.0
13.4
0.4
0.4
0.3
0.4
0.3
0.4
0.4
0.3
0.4
0.2
Figure 1. HPLC chromatograms of samples containing various potential
interfering agents including lumiflavin, FMN, FMF and FAD.
Isolated fractions were analyzed using the diode array detector to identify
UV maxima in the individual peaks. Samples were then forwarded to
Cardinal Health (Raleigh, NC) for HPLC-MS/MS analysis. They were
protected from light and shipped at room temperature. For analysis, samples
were transferred to dark glass vials at room temperature. Analysis of all
samples was performed using a Cohesive Technologies 2300 HPLC system
coupled to a Sciex API3000 triple quadrupole mass spectrometer.
Extractions of each of the unknown solutions were performed on a Cohesive
Cyclone-P 1 3 50 mm extraction column and on either a 250 3 4.6 mm
cyano or a 150 3 4.6 mm cyano analytical column for analytical
separations. All mass spectrometric analysis was done using electrospray
negative ionization. In cases where more signal was needed, samples were
concentrated using a Turbo-vap at 308C with nitrogen gas. The instrument
was protected from light during the evaporation. For all injections, the
following MS/MS transitions were scanned, based on analysis of standards:
RB, 375.1 . 255.1 amu; KF, 373.1 . 241.3 amu; LC, 240.9 . 198.1 amu;
FMF, 282.9 . 240.2 amu.
Apheresis platelet preparation. Single donor platelets (a minimum of 270
mL) were collected by an accredited blood bank facility using a Gambro
BCT Inc. TRIMAÒ Automated Blood Component Collection System. The
platelet product was held between 2 and 30 h in the TRIMA collection bag
before subsequent processing. The bag containing the platelets was
connected to the pathogen reduction illumination–storage bag (ELPÔ)
using a TerumoÒ Sterile Tubing Welder. After sterile connection, 250 6 5
mL of platelet product was transferred into the illuminator bag, which
contained 28 mL of RB solution (500 lM). The transfer tubing was sealed
using a SebraÒ hand-held, radio frequency tubing sealer. After connection,
the two bags were separated and the original collection bag was discarded.
Each final platelet product to be treated contained 1260 3 103 to 1690 3 103
platelets/lL suspended in approximately 278 mL of 90% autologous
plasma in a 1 liter citrate-plasticized, polyvinyl chloride ELP bag with an
illumination surface area of 347 cm2/side.
Plasma derived from buffy coats. Buffy coat platelets were prepared from
whole blood that was stored at room temperature for 16–24 h after
collection. The platelets were spun down at 16 100 RCF for 3 min and
diluted 1:50 with 0.9% saline before analysis.
The PRT process. A complete description of the procedures used for this
process has been described previously (Ruane et al.). Briefly, the platelet
concentrates were treated separately with 6.2 J/mL of light. The lamp
phosphor possesses an output range from 265 to 370 nm. The platelet
product was placed in a product chamber where mixing (on a motorized
platen) and exposure to light took place. Total illumination ranged from
8 to 10 min. Dedicated fans were used to cool each lamp chamber and
product chamber. Product temperatures during illumination ranged from
228C to 248C.
326
Purification of LC. The average purity of reagent LC (Aldrich PN
10,321-7) was found to be approximately 68–85% as determined by
negative ion mass spectroscopy and HPLC. LC was purified before use as
an analytical standard as follows: reagent grade LC (250 mg) was added to
a 250 mL amber volumetric flask and diluted to the mark with reagent grade
water. The solution was sonicated (VWRÒ sonicator model 150HT) at 808C
for 2 h and then vacuum filtered while hot, using a 0.45 lm filter. The filter
cake, which consisted of purified LC, was dried at 1058C for 2 h (the
impurity is soluble in water and LC is relatively insoluble). The LC purity
after this process was found to be greater than 99%. LC purity was
confirmed using mass spectrometry, proton nuclear magnetic resonance (1H
NMR), HPLC and elemental analysis. An API3000 triple quadrupole mass
spectrometer using negative ionization mode observed the parent M1 ion
241 m/z concurrent with the LC nominal molecular weight of 242 g/mol in
the purified sample. Elemental analysis—found: C 59.04, H 3.81, N 22.95;
required: C 59.50, H 4.16, N 23.13. 1H NMR (D2O), 2.39, s, 3H; 2.41, s,
3H; 7.61, s, 1H; 7.78, s, 1H.
Preparation of FMF reference compound. FMF was synthesized
according to the method of Fall and Petering and characterized using
NMR and mass spectrometry (35).
Calibration standard preparation. Analytical standards were prepared by
diluting a stock solution of 50 lM RB and 50 lM LC in saline. The stock
solution was prepared by adding LC (12.1 mg) to a 1 liter amber volumetric
flask and adding approximately 950 mL of pH 5.0 saline. The solution was
sonicated using a VWR sonicator (model 150HT) at 70–1008C for
approximately 2 h until all LC was in solution. RB (18.8 mg) was then
added and the solution allowed to cool while stirring overnight. It was then
filled to the mark with pH 5.0 saline. Calibration standards were prepared
fresh every 24 h.
HPLC validation for quantitative analysis of RB and LC. Validation of
the method for RB and LC, the principal photoproduct of RB was carried
out during an 8 day period. Both these agents were present in sufficient
quantities in blood products to validate the methodology. Other components
(FMF, 2KF, 4KF) were isolated by bulk HPLC and characterized by mass
spectroscopy and UV–visible (UV-VIS) spectroscopy. Results from these
studies were used to determine concentrations of these agents in blood
components, but validation of the methodology for quantitative determination of these components was not performed. Linearity of the calibration
curve was 0.999 (r2) or better for all 8 days of validation. For five replicates
of both RB and LC at three concentrations (25, 37.5 and 50 lM), the
average relative standard deviations were RB 5 0.64% and LC 5 0.76%.
For these 30 samples, the average percent recovery for RB was 97%
whereas the average percent recovery for LC was 102%. The limit of
quantitation for RB (undiluted) was 16 nM and for LC was 60 nM. Samples
were diluted 1:50 with 0.9% saline pH 5.0 before analysis. The range of the
method for RB is 0.016–1.500 lM and for LC 0.06–1.500 lM. A typical
analysis involved two replicates of each sample. No loss of peak shape,
peak height or retention time was noted for runs using as many as 15
samples (30 samples when replicates are accounted for).
During the 8 day validation, all retention times for RB samples were
within 7.8 6 0.2 min and all retention times for LC samples were within
612
Christopher C. Hardwick et al.
Table 2.
RB and photoproducts characteristics
Compound
Average HPLC
retention
time (min)
RB
LC
2KF
8.4
15.5
9.1
4KF
9.9
FMF
10.8
UVmax (nm)
223,
218,
224,
446
222,
446
222,
267, 374, 444
260, 352
268, 372
268, 374
268, 372, 446
Table 3.
MS/MS
transitions
(amu)
375.1
240.9
373.1
373.1
373.1
373.1
282.9
.
.
.
.
.
.
.
255.1
198.1
241.3
255.1
241.3
255.1
240.2
15.4 6 0.2 min. The average resolution was 10.1 and the average tailing
was 0.34, passing the Food and Drug Administration validation requirements of 2.0 resolution and 2.0 tailing (36).
Reinjection precision analysis was performed to determine whether an
analytical run could be restarted in case of instrument failure. Analysis was
performed on a calibration curve and on 30 samples, 10 each at three
different concentrations. The calibration curve was followed by 30 samples,
including blanks and check standards every 10 samples, simulating a routine
analysis. After completion of the sequence, the entire sequence was
restarted. The difference in measured RB and LC concentrations from the
first run and second run was less than 61.5%.
Interference. Several analytes were run to test for interference including
FAD, FMN, lumiflavin and FMF. None of these analytes interfered with the
analysis of RB and LC. Results from sample runs with these agents are
included in Fig. 1.
RB and photoproduct analysis in platelet concentrates. Ten platelet
concentrates spiked with 50 lM RB were treated with 6.2 J/mL light. The
concentrations of RB, LC and other photoproducts were measured before
and after spiking with RB, after illumination and after 5 days of storage on
a Helmer shaker, respectively (22–248C, shaker speed of 72 6 5 cpm). All
samples were stored in the dark until analysis, whereupon a 4 mL sample
was drawn from each platelet product. Each sample was centrifuged at 3000
RCF for 10 min to remove the cellular components. The supernatant was
respun at 16 100 RCF for 3 min and the supernatant diluted 1:50 with 0.9%
saline. The samples were then placed in amber HPLC vials for analysis.
Analysis included characterization of peaks associated with the 2KF, 4KF
and FMF as well. Quantification of these latter samples was conducted on
the basis of extinction coefficients for the parent molecule and HPLC
analysis. Identification of peaks associated with these agents was performed
through analysis of isolated fractions by mass spectroscopy–HPLC and
UV-VIS absorption characteristics (Table 2). In the case of FMF, direct
comparison with a de novo synthesized standard was possible (Table 3).
Additional samples taken from normal donors were analyzed for the
presence of RB and RB catabolites or photoproducts. These samples were
assayed directly without any RB addition to determine the levels of each
component present in a natural state in blood components.
RESULTS
RB, LC, 2KF, 4KF and FMF analysis in apheresis platelets
The level of RB and LC and other catabolites of RB in the platelet
products after spiking with 50 lM RB are listed in Table 4. The
average RB concentration after the addition of 28 mL of 500 lM
RB to 250 6 5 mL platelet product was 48.3 lM (n 5 10, Table 4).
PRT treatment resulted in 78.9% (n 5 6) recovery of the RB
originally present after dilution and before illumination. An additional 6.2% of this initial level of RB was recovered in the form of
LC, resulting in a total of 85.1% recovery of the initial level of RB.
The remaining levels were identified to be composed of FMF, 2KF
and 4KF. Separation of these components by bulk HPLC was
conducted to isolate purified fractions of each. These samples were
examined to obtain mass spectra data and UV-VIS spectra characteristics. Upon storage for 5 days, the amounts of RB and LC both
FMF standard versus isolated compound
Compound (FMF)
Retention
time (min)
UVmax
Standard reference
10.8
224, 268, 372, 446
Isolated compound
10.8
222, 268, 372, 446
Mass spectra
ions(m/z)
283
240
283
240
(parent),
(daughter)
(parent),
(daughter)
increased by a measurable extent (Fig. 2b,c). A total of 80.8% RB
(n 5 10), 7.4% LC, 1.4% 2KF, 0.3% 4KF and 3.5% FMF were
recovered (n 5 10), yielding a total mass balance of 94.4% after 5
days of storage. These results are given in Table 4. The HPLC
chromatograms of the PRT-treated platelets before illumination,
after illumination and after 5 days of storage are shown in Fig. 2.
Quantitative analysis of endogenous RB and
photoproducts–catabolites in plasma
A modified version of the current method was used to measure
endogenous concentrations of RB and RB catabolites in plasma.
The injection volume was increased from 10 to 100 lL and the
dilution with saline was reduced from 1:50 to 1:5. This gave a 100fold increase in RB detection limits. A six-point calibration curve
was run on the same day of analysis using standards ranging from
1 to 50 nM (1, 2, 5, 10, 20 and 50 nM). Thirty samples of plasma
derived from buffy coat platelets were analyzed for RB and its
photoproducts–catabolites. The average concentration of RB was
23.9 nM (range 8.6–79.6 nM) (see Fig. 3). Excellent results were
obtained with this adapted HPLC method. Blanks were run before
and after the calibration curve and between each of the 30 samples.
RB was not detected in any of the blanks. The linear regression of
the calibration curve was greater than 0.999. Levels of LC and
other photoproducts–catabolites of RB present in these products
varied as shown in Table 5. Out of 30 products in total analyzed
using this method, all samples demonstrated measurable levels of
RB. Only 23 of the 30 products demonstrated measurable levels of
LC. A total of three of the 30 had measurable levels of all four
major photoproducts. These also corresponded to those samples
having the highest concentrations of RB. The average concentrations determined for each of these products as well as the ranges
observed are listed in Table 5.
DISCUSSION
The methodology described represents a sensitive HPLC method
for the quantification of RB, LC, 2KF, 4KF and FMF in blood
components that does not require a protein precipitation or organic
solvent extraction step. The method simply removes the cellular
components by centrifugation followed by dilution with saline and
analysis using a reverse phase cyano-column (ZorbaxÒ 80Å SBCN, 4.6 3 250 mm, 5 micron). We have found that the low concentration of protein after dilution does not interfere with the assay.
Typically, C18 reversed phase columns are used to analyze RB
and related compounds in aqueous solutions (15,18–20,22,24–
29,31–34, 36). However, we found that the use of this stationary
phase was not satisfactory and separation of photoproducts from RB
after photolysis was not feasible. A reverse phase cyano-column was
found to be optimal in resolving compounds closely eluting to RB
(Fig. 2). Other columns were used and separation was not optimal
327
Photochemistry and Photobiology, 2004, 80
613
Table 4. RB and photoproducts in apheresis platelets*
RB
% of initial
LC
% of initial
2KF
% of initial
4KF
% of initial
FMF
% of initial
Totals
PRE (lM) POST (lM)à
POST 5 days (lM) 48.33 6 1.13 (47.1–50.4)
38.68 6 0.68 (38.0–39.9)
78.9
2.90 6 0.20 (2.5–3.0)
6.2
1.27 6 0.17 (1.10–1.49)
2.57
0.53 6 0.03 (0.48–0.58)
1.06%
1.99 6 0.24 (1.63–2.32)
4.02
45.4 lM; 93.4%
39.65 6 1.22 (37.0–41.3)
82.0
3.60 6 0.30 (3.2–4.1)
7.5
0.71 6 0.12 (0.59–0.90)
1.44
0.16 6 0.04 (0.11–0.22)
0.32%
1.72 6 0.22 (1.42–2.07)
3.49
45.8 lM; 94.4%
0.23 6 0.08 (0.1–0.3)
N/A
N/A
N/A
*PLT, apheresis platelet product before spiking with 50 lM RB; PRE, after spiking (50 lM RB); POST, after treatment; POST 5 days, after a further 5
days of storage.
n 5 10.
àn 5 6. Values in parentheses are the range.
(Agilent (Zorbax) Columns: SB-C18, XDB-C18, 300 Extend C18,
300 SB-C8, XDB-C8, SB-C8, 300SB-C3, 300SB-CN, Carbohydrate, SB-Aq, Eclipse AAA, NH2, Diol and GF-250).
The measurement of vitamin B2 in blood components is not new
(15,16,19–24,32). Typically, FAD in the blood is converted to the
more stable FMN. Free RB and FMN are then measured after their
extraction into organic solvents. These methods also require the
acidic (usually TCA) precipitation of blood proteins before analysis.
This has one significant drawback. RB and related compounds tend
to coprecipitate with the protein making accurate determinations
difficult. In most cases, although agents such as FMF, 2KF and 4KF
have been previously identified, their low levels in naturally
occurring blood products and the low sensitivity inherent in
previous methodologies prohibited their routine identification and
quantification in blood components. The increased sensitivity of the
method described in this study permits this direct analysis.
The method described in this article has been developed to
support a PRT known as MIRASOL PRT. The technology involves
the inactivation of pathogens in platelet concentrates using 50 lM
RB and light. During the course of this study, we measured the
Figure 2. RB (50 lM, diluted 1:50 with 0.9% saline, pH 5.0) in apheresis
platelets illuminated 6.2 J/mL with UV. (a) Before illumination. (b) After
illumination (RB 5 78.9%, LC 5 6.2%). (c) After illumination and 5 days
storage (RB 5 82.0%, LC 5 7.5%). a, RB; b, mass spectrum and UV-VIS
are consistent with 2KF; c, mass spectrum and UV are consistent with 4KF;
d, mass spectrum and UV are consistent with FMF; e, LC.
328
amount of RB, LC, FMF, 2KF and 4KF in 10 apheresis platelet
products before and after treatment. The platelet concentrates were
also stored at 22–248C on a Helmer shaker for 5 days, whereupon
the amounts of each of these agents were reanalyzed. We were able
to accurately determine the amount of RB converted to photoproducts during the process. A total of 78.9% RB remained
immediately after irradiation. This amount increased upon 5 days of
storage to 82.0%, presumably because of photoproducts (not LC)
reverting back to RB during this period. The amount of LC also
increased upon storage from 6.2% to 7.5%. The total mass balance
of both compounds on Day 5 was 89.5%. The remaining 10.5% is
made up of other photoproducts that have been positively identified
and quantified through mass spectroscopic and other analytical
methods. Figure 2 shows an overlay of HPLC chromatograms of
RB in a diluted platelet product (1:50), before illumination (a), after
illumination (b) with the PRT process and Day 5 after treatment (c).
The photoproducts b and c have been identified as 2KF and 4KF,
respectively. Their UV-VIS absorbance spectra (both are nearly
identical to that of RB) and mass spectra are consistent with this
hypothesis. Results from HPLC, UV-VIS analysis and mass spectra
data are depicted in Table 2. FMF (d) has been positively identified
by comparison with an authentic sample. Results from this analysis
are depicted in Table 3. Quantification of these photoproducts has
been conducted using the extinction coefficients for the parent
molecule and are included in Table 4.
Figure 3. Endogenous concentration of RB in 30 plasma samples derived
from buffy coat platelets. Average 5 23.9 nM.

Cerus Corporation INTERCEPT Blood System for plasma

Transcription

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